APPLICATIONS OF RADIOTRACER IN PLANT BIOLOGY
_______________________________________
A Dissertation
Presented to
The Faculty of the Graduate School
University of Missouri-Columbia
_______________________________________________________
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
_____________________________________________________
by
LIHUI SONG
Dr. Silvia Jurisson and Dr. Gary Stacey, Dissertation Supervisors
December 2013
The undersigned, appointed by the dean of the Graduate School, have examined the
dissertation entitled
APPLICATIONS OF RADIOTRACER IN PLANT BIOLOGY
presented by Lihui Song,
a candidate for the degree of [doctor of philosophy of Chemistry],
and hereby certify that, in their opinion, it is worthy of acceptance.
Professor Silvia Jurisson
Professor Gary Stacey
Professor David Robertson
Professor Timothy Glass
Professor Richard Ferreri
To My loves,
I would like to dedicate this work to my dear family who have been loving me
and supporting me unconditionally, even though I have not spent any New Year eves
with them during the last five years. I also would like to dedicate this work to my loved
fiancé, Noah Marchal, for his understanding, support, patience and help with revision
during the dissertation writing process.
ACKNOWLEDGEMENTS
There are so many people who helped me create new neuron connections in my
brain throughout my graduate career and all of them deserve more than just the ink on
one page of my dissertation.
First, I would like to thank my advisor, Dr. Silvia Jurisson, and my co-advisors, Dr.
Gary Stacey and Dr. Richard Ferreri, for their guidance, patience, encouragement and
revisions on my dissertation. Gary led me to the door of this research opportunity, Silvia
welcomed me in, and Rich taught me hand by hand how to conduct radiotracer studies on
plants. Without them, I would not have gained the knowledge and experience I had today
and figured out what I love to do for my future career. I also would like to thank my
dissertation committee members, Dr. Timothy Glass and Dr. David Robertson for their
continuous support and their help with the revision of my dissertation. Another person I
would like to thank here is Minviluz (Bing) G. Stacey. Although she is not in my
dissertation committee, she taught me how to conduct plant biological studies and other
important molecular biology techniques, especially helped me with the study mentioned
in Chapter 2.
In addition, I would like to thank Dr. Jurisson’s Group, Dr. Stacey’s Group and Dr.
Ferreri’s Group. Some of them helped me a lot with experimental design and actual
experiments, and some of them are my spiritual support. I have enjoyed every one of
their companies during my graduate career.
There are also facilities I would like to acknowledge for conducting analyses for
my work. They are MU Research Reactor (David J. Robertson, James Guthrie and Gang
Li) and Environmental Molecular Sciences Laboratory/ Pacific Northwest National
ii
Laboratory (EMSL/PNNL; Lizabeth Alexander) for the ICP-MS analyses; MU Molecular
Cytology Core (Yan Liang) for providing the microscopic imaging services.
At last, I would like to thank MU Chemistry Department and Department of
Energy Biological and Environmental Research (DOE-BER) program for the financial
supports.
iii
Table of Contents
Acknowledgments ...................................................................................................................... ii
List of Tables ........................................................................................................................... viii
List of Figures ............................................................................................................................. x
List of Schemes ........................................................................................................................ xiii
List of Equations .....................................................................................................................xiv
Abstract ...................................................................................................................................... xv
Chapter 1: Introduction
1.1 General Concepts of Radiotracer ...................................................................................... 1
1.2 Techniques used for Radiation Detection ....................................................................... 3
1.3 Advantages and Disadvantages of Radiotracer Studies ................................................ 6
1.4 Underlying Assumptions in Design of Radiotracer Experiments ............................... 6
1.4.1 Basic Assumptions ...................................................................................................... 6
1.4.2 Evaluation of the Feasibility of Radiotracer Experiments ..................................... 8
1.5 History of Plant Biological Studies using Radioactive Isotopes ................................. 9
1.6 Radiotracer Studies in This Dissertation ...................................................................... 10
References ............................................................................................................................... 12
Chapter 2: Accumulation of Heavy Metals by the Arabidopsis opt3-2 Mutant
2.1 Isotope Summary ............................................................................................................. 14
2.2 Background ....................................................................................................................... 14
2.3 Materials and Methods .................................................................................................... 16
2.3.1 Materials .................................................................................................................... 16
2.3.2 Plant Growth Conditions ......................................................................................... 16
iv
2.3.3 Determination of Chlorophyll Content .................................................................. 18
2.3.4 ICP-MS Analysis ...................................................................................................... 18
2.3.5 Radioactive 203Pb2+ Uptake Assay and Imaging .................................................. 19
2.3.6 Yeast Transformation and Growth Assays .......................................................... 19
2.3.7 Statistic Analysis ...................................................................................................... 20
2.4 Results ............................................................................................................................... 20
2.4.1 Toxic Effects of Heavy Metals on Plant Growth ................................................. 20
2.4.2 Heavy Metal Quantification in Plant Tissues by ICP-MS .................................. 21
2.4.3 Radioactive 203Pb2+ Uptake Assay ......................................................................... 22
2.4.4 IRT1 does not Mediate Pb or Hg Uptake .............................................................. 22
2.5 Discussion ......................................................................................................................... 23
References ............................................................................................................................... 33
Chapter 3: Dynamics of Sugar Metabolism and Re-location in Arabidopsis Starch
Mutants Using 11CO2
3.1 Isotope Summary ............................................................................................................. 36
3.2 Background ....................................................................................................................... 36
3.3 Materials and Methods .................................................................................................... 41
3.3.1 Materials .................................................................................................................... 41
3.3.2 Plant Growth Conditions ......................................................................................... 41
3.3.3 Starch Staining Assay .............................................................................................. 42
3.3.4 11CO2 Production, Pulsing and Incubation Apparati ........................................... 42
3.3.5 Tissue Extraction and Soluble Sugar Analysis ..................................................... 43
3.3.6 Carbon Allocation .................................................................................................... 45
3.3.7 Phosphor Imaging .................................................................................................... 46
3.3.8 Root Morphology ..................................................................................................... 46
v
3.3.9 Root Elongation and Gravitropic Response .......................................................... 46
3.3.10 Statistical Analysis ................................................................................................. 46
3.4 Results ............................................................................................................................... 47
3.4.1 Plant Growth ............................................................................................................. 47
3.4.2 Plant Physiology ....................................................................................................... 47
3.4.3 Carbon Metabolism .................................................................................................. 49
3.4.4 Root Gravitropic Response ..................................................................................... 50
3.5 Discussion ......................................................................................................................... 51
3.5.1 Carbon Metabolism and Allocation ....................................................................... 51
3.5.2 Root Morphology and Gravitropic Response ....................................................... 54
References ................................................................................................................................... 65
Chapter 4: Effect of Fe Statuse on Carbon Metabolism in Arabidopsis
4.1 Isotope Summary ............................................................................................................. 69
4.2 Background ....................................................................................................................... 69
4.3 Materials and Methods .................................................................................................... 72
4.3.1 Materials .................................................................................................................... 72
4.3.2 Plant Growth Conditions ......................................................................................... 72
4.3.3 Chlorophyll Content Determination ...................................................................... 73
4.3.4 Proton Concentration Measurements in the Rhizosphere ................................... 74
4.3.5 59Fe Uptake Assay .................................................................................................... 74
4.3.6 11CO2 Production, Pulsing and Incubation Apparatus ......................................... 75
4.3.7 Tissue Extraction and Soluble Sugar Analysis ..................................................... 76
4.3.8 Carbon Allocation .................................................................................................... 78
4.3.9 Phosphor Imaging .................................................................................................... 79
3.3.10 Statistical Analysis ................................................................................................. 79
vi
4.4 Results ............................................................................................................................... 79
4.4.1 Plant Growth ............................................................................................................. 79
4.4.2 Acidification of Rhizosphere through Root Exudation ....................................... 80
4.4.3 59Fe Uptake under Various Levels of External Fe Availability .......................... 81
4.4.4 Carbon Fixation, Metabolism and Allocation ...................................................... 81
4.5 Discussion ......................................................................................................................... 83
References ................................................................................................................................... 97
Chapter 5: Conclusions and Future Studies .....................................................................101
References .................................................................................................................................104
Appendix A: Supplementary Data ..................................................................................... 105
Appendix B: Protocols ..........................................................................................................140
Vita .............................................................................................................................................146
vii
List of Tables
Page
Table 2-1.
Fractions of total 203Pb taken up in the plant transported into roots and
shoots of the WT (col-0) and the opt3-2 mutant .............................................. 32
Table A1-1. Heavy metal treatments tested on the opt3-2 mutant plants ......................... 105
Table A1-2. Experimental parameters for 35S-glutathione uptake assay ......................... 118
Table A1-3. Data of root / shoot growth of plants under Hg or Pb treatments ............... 119
Table A1-4. Data of chlorophyll content .............................................................................. 120
Table A1-5. ICP-MS Pb data (ppb)........................................................................................ 121
Table A1-6. ICP-MS Hg data (ppm) ...................................................................................... 122
Table A1-7. 203Pb distribution in opt3-2 mutant and WT plants ....................................... 123
Table A2-1. List of sugars ...................................................................................................... 124
Table A2-2. Data of Chlorophyll content ............................................................................. 125
Table A2-3. Comparison of root elongation among three starch mutants and
wild type Arabidopsis seedlings ...................................................................... 126
Table A2-4. Comparison of root gravitropic responses among three starch mutants
and wild type Arabidopsis seedlings ............................................................... 127
Table A2-5. Percentage of 11C sugar partitioning in the starch mutants and the
wild type ............................................................................................................. 128
Table A2-6. 12C sugar partitioning in the starch mutants and the wild type .................... 129
Table A2-7. Percentage of total fixed 11C assimilated into insoluble carbohydrates ..... 130
Table A2-8. 11C-carbohydrate relocation to belowground and root exudates ................. 130
Table A3-1. Chlorophyll content ........................................................................................... 131
Table A3-2.
59
Fe uptake under various levels of external Fe availability ....................... 132
Table A3-3. Total 11CO2 fixation .......................................................................................... 133
viii
Table A3-4. 11C-sugar partitioning in the leaves of three types of Arabidopsis
seedlings ............................................................................................................. 137
Table A3-5. 11C-carbohydrate relocation to belowground and root exudates ................. 138
ix
List of Figures
Page
Figure 2-1. Shoot growth in different heavy metal treatments ......................................27
Figure 2-2. Root growth in different heavy metal treatments ........................................28
Figure 2-3. ICP-MS shows the amount of heavy metals in different above-ground
tissues of both col-0 and opt3-2 mutant ......................................................29
Figure 2-4. Comparison of different yeast transformants grown under various
concentrations of heavy metal treatments ...................................................30
Figure 2-5. Phosphor image of 203Pb distribution in opt3-2 mutant and wild type
plants ...........................................................................................................31
Figure 2-6.
203
Pb distribution in opt3-2 mutant and wild type plants ............................31
Figure 3-1. Brief summary of carbon fixation and downstream carbon metabolic
pathway .......................................................................................................56
Figure 3-2. The apparati used for 11CO2 pulsing and incubation ...................................57
Figure 3-3. Comparison of root/shoot growth and chlorophyll content among
the three starch mutants and WT ................................................................58
Figure 3-4. Comparison of root elongation and gravitropic responses among three
starch mutants and wild type Arabidopsis seedlings ..................................59
Figure 3-5. Carbon-11 phosphor imaging of the three starch mutants and the
wild type ......................................................................................................60
Figure 3-6. Iodine staining assays of starch in three starch mutants and wild type
Arabidopsis ..................................................................................................60
Figure 3-7. Iodine staining assay of starch in the root tips of three starch mutants
and wild type Arabidopsis ...........................................................................61
Figure 3-8.
11
C-carbohydrate relocation to belowground and root exudation ...............62
Figure 3-9.
12
C and 11C sugar partitioning in the starch mutants and the wild type ......63
Figure 3-10. Percentage of total fixed 11C assimilated into insoluble carbohydrates ......64
x
Figure 4-1. Two strategies for iron uptake in plants ......................................................89
Figure 4-2. Experimental set-up for 59Fe uptake ............................................................75
Figure 4-3. Comparison of root/shoot growth and chlorophyll content among the
mutants and WT ..........................................................................................90
Figure 4-4. Measurement of root exudate pH of Arabidopsis seedlings (wt, opt3-2 and
irt1-1) grown under various concentrations of Fe treatments using a
pH indicator ................................................................................................91
Figure 4-5.
59
Fe uptake under various levels of external Fe availability .......................92
Figure 4-6. Total 11CO2 fixation .....................................................................................93
Figure 4-7.
11
C-sugar partitioning in the leaves of three types of Arabidopsis
seedlings .......................................................................................................94
Figure 4-8. Allocation of 11C-photosynthates to the belowground ................................95
Figure 4-9. Root exudation ............................................................................................96
Figure 5-1. A conceptual model used to interpretate 12C and 11C data ........................102
Figure A1-1. Comparisons of seed germination rates under different heavy metal
treatments within the first three days .......................................................108
Figure A1-2. Comparisons of root elongation under different heavy metal
treatments .................................................................................................108
Figure A1-3. 1H-NMR of RBS (300 MHz), dissolved in CDCl3....................................111
Figure A1-4.
13
C-NMR. of RBS (500 MHz), dissolved in CDCl3.................................112
Figure A1-5. Fluorescence Spectra of RBS with different concentrations of Hg2+ ........113
Figure A1-6. Comparison of shoot growth of the opt3-2, irt1-1 mutants and the
wild type Arabidopsis with various concentrations of Pb/Hg
treatments .................................................................................................114
Figure A1-7. Comparison of the root growth for the opt3-2, irt1-1 mutants and the wild
type Arabidopsis under various concentrations of Pb/Hg treatments ......115
Figure A1-8. Illustration of load leaf incubation in the [35S]-glutathione containing
solution ......................................................................................................116
Figure A1-9. 35S-glutathione uptake through leaf administration and distribution patterns
in the opt3-2 mutant (right) and the WT (left) ..........................................117
xi
Figure A2-1. Carbon fixation in the three starch mutants and the wild type
Arabidopsis ..............................................................................................125
Figure A3-1. Total 11CO2 fixation. The experiments were conducted both in the
morning (A) and afternoon (B) .................................................................133
Figure A3-2. 11C-sugar partitioning in the leaves of three types of Arabidopsis
seedlings (Fructose) .................................................................................134
Figure A3-3. 11C-sugar partitioning in the leaves of three types of Arabidopsis
seedlings (Glucose) ...................................................................................135
Figure A3-4. 11C-sugar partitioning in the leaves of three types of Arabidopsis
seedlings (Sucrose) ...................................................................................136
xii
List of Schemes
Page
Scheme A1-1. Structure of RBS ............................................................................................ 109
Scheme A1-2. Possible reaction mechanisms of RBS with HgCl2 .................................... 110
xiii
List of Equations
Page
Equation 3-1: Sucrose synthesis and degradation ................................................................ 37
Equation 3-2: Biological functions of the enzyme ADP-glucose pyrophosphorylase
(AGP) and phosphoglucomutase (PGM) ...................................................... 38
xiv
ABSTRACT
A radioactive tracer (radiotracer) is generally defined as a radioactive isotope that
is used as a tracer, which can be followed or tracked within a system of interest. The use
of radiotracers involves the substitution of a radioactive isotope for one of the naturally
occurring isotopes of a particular element. Radiotracers have a wide range of application
due to two unique features: a high level of detection sensitivity, and an ability to integrate
into living systems. These features make the use of radiotracers particularly useful for
studying the dynamic processes that comprise metabolic activity. This work focuses on
the use of radiotracers to identify and follow specific biological pathways to facilitate our
understanding of plant biology. All of the plants used in this work are Arabidopsis
thaliana plants (wild type, col-0 and its different mutants).
Plants are the most common living organisms on earth and have critical roles for
the global environment, including human societies. Plants are able to reduce the problem
of pollutions, such as uptake contaminants from soils and waters. Also, plants are the
basic food producers for other living organisms, which are not only the ones above
ground, but also belowground in the rhizosphere (microorganisms). Therefore, if the
plant systems are disturbed, it could affect the ecosystem dramatically. In this dissertation,
we are trying to use radiotracers to explore the mechanisms of the basic physiology and
metabolisms in plants.
In Chapter 2, heavy metal uptake and accumulation in one of the Arabidopsis
mutant, opt3-2 mutant, was investigated in order to understand the possible mechanisms
of phytoremediation. The results showed that the opt3-2 mutant plants accumulate Pb2+ in
xv
both influorescence stems and rosette leaves compared to the wild type (WT). In addition,
the results from the radioactive
203
Pb uptake assays indicated that the heavy metal
accumulation phenotype in the opt3-2 mutant plants is not due to a kinetic rapid uptake,
but to a long-term regulation.
In Chapter 3, the carbon metabolism and translocation in Arabidopsis plants were
investigated using both
12
C and
11
C methods to explore the dynamics of carbon flux in
plants, as well as diurnal effects on carbon flux. Three starch mutants (sex1-1, adg1-1 and
pgm-1) and the WT Arabidopsis were used in this study. The results showed that starch
regulation is essential not only for plant growth, but also affects sugar metabolism
(fructose, glucose and sucrose), carbon allocation to plant roots, and root exudation.
In Chapter 4, the relationship between Fe status and carbon metabolism was
discussed. Both
12
C and
11
C methods were used in this study to investigate the carbon
metabolism and translocation in Arabidopsis plants. Two iron-transport mutants (opt3-2
and irt1-1 mutants) and the WT Arabidopsis were used for this study. The results showed
that the Fe status in plants affects the carbon fixation ability in plant leaves, and also
alters carbon partitioning (possibly through the production of more organic acids under
Fe deficient stress), carbon allocation, and root exudation in plants. Diurnal effects on
carbon metabolism and allocation were observed in this study, as well.
xvi
Chapter 1: Introduction
1.1 General Concepts of Radiotracer
A radioactive tracer (radiotracer) is generally defined as a radioactive isotope
used as a tracer, which can be followed or tracked in a system of interest. The use of
radiotracers involves substituting a radioactive isotope for one of the naturally occurring
isotope of a particular element (this could be chemically bound or isolated as an ion).
Radiotracers are used in a wide range of areas, such as the basic sciences, medical
sciences and in industry. For example, radiotracers are commonly used in pharmaceutical
research to monitor drug metabolism in vivo. Radiotracers can also be used as diagnostic
reagents to locate cancerous tumors in vivo or therapeutic reagents to kill the cancerous
cells. This dissertation will thoroughly discuss how radiotracers can identify or follow
specific biological pathways to aid in understanding plant biology.
Radioactive isotopes spontaneously decay through different modes to form more
stable nuclei. There are several major decay modes: alpha decay, beta decay, positron
decay, electron capture decay and gamma decay.
Alpha (α) decay occurs among the elements with higher atomic numbers,
especially with Z > 82. In this decay mode, an α particle (He2+) is emitted from the
radioactive nucleus. Alpha particles cannot travel far due to their large size and high
charge. Alpha particles lose energy to the surrounding matters through ionizing
interactions, which can cause damage to the tissues. Thus, α-emitting radioisotopes are
not generally used in radiotracer studies.
1
Chapter 1
Beta (β-) decay generally occurs in nuclides with an excess of neutrons. βparticles (or negatrons) are high energy electrons emitted from the nucleus, which can
travel further than α particles due to their lower mass. The ability of β- particles to
penetrate materials makes them easier to detect by radiation detectors, such as liquid
scintillation detectors or gas ionization detectors.
Positron (β+) decay occurs in proton-rich nuclides. β+ particles are equal and
opposite to β- particles (e.g., they have the same mass as β- particles, but are positively
charged). A β+ particle is the antiparticle of an electron and will annihilate with a nearby
negatively charged electron to form two coincident 511 keV photons whose paths are
approximately
apart from each other.
Another type of decay that often occurs in proton-rich nuclides is electron capture
decay, which is a decay mode in which one of the inner orbital electrons interacts with
the nucleus, combining with a nuclear proton to form a neutron. X-rays and Auger
electrons are often emitted following electron capture decay as a consequence of the
rearrangement of the orbital electrons.
In gamma (γ) decay, the nucleus releases its excess excitation energy by the
emission of a photon(s). This decay mode can occur along with other types of decay
modes or by itself. For example, a metastable isotope decaying to its ground state via
isomeric transition will only emit γ-rays.
2
Chapter 1
1.2 Techniques used for Radiation Detection
Because radioactive nuclei in radiotracers decay by emitting different types of
radiation (particles and/or photons), choosing the appropriate detection techniques is
essential to accurately quantify or demonstrate the distribution of the radiotracers in the
system(s) of interest.
Common Radiation Detectors
To quantify the amount of radiotracers present, counting the activity of a sample
(such as plant tissues) is generally used. There are several types of radiation detectors,
which make use of different detection mechanisms. Gas ionization detectors and
scintillation detectors are commonly used radiation detectors.
Gas ionization detectors take advantage of the ionizing effect of radiation on
gasses, which subsequently generates electrical current that is used as a signal indicating
the radiotracer activity. The Geiger-Muller (G-M) detector is a widely used gas ionization
detector, which is highly efficient in detecting α and β- particles but only 1% efficient in
detecting γ rays due to their high penetrating ability through low density gases. The main
difficulty in detecting α or low-energy β- radiation is the extent of absorption occurring in
the detector window.
In scintillation detectors, a portion of the energy of ionizing radiation is
transferred to fluor atoms or molecules (which can give rise to fluorescence) in a
crystalline solid or liquid cocktail. The absorbed energy causes excitation of orbital
electrons in the fluor. De-excitation gives rise to the emission of the absorbed energy as
3
Chapter 1
electromagnetic radiation in the visible or near ultraviolet region (scintillation). Although
observing weak scintillations visually is possible, this is usually not a feasible detection
method. In general, the photons generated in the scintillation process are converted to
photoelectrons and then amplified to form a detectable electrical pulse using a
photomultiplier. Therefore, during this process, the original radiation energy is
transformed into a measurable pulse. Scintillation detectors generally have two types,
solid scintillation (SS) and liquid scintillation (LS). The commonly used SS detector is
the NaI (Tl) detector, which has a high efficiency for γ detection. The LS detector uses a
solvent cocktail (contains fluor molecules), which directly interacts with the sample.
Because the cocktail and sample are in contact with one another, energy is not lost as
much as that in the SS detectors by the ionizing radiation while passing through the
detector window as in the SS detector, which makes it more efficient for α and βparticles, especially low energy β- particles (e.g., β- particles emitted from biologically
useful isotopes like 3H,
14
C, and
32
P). However, color and chemical quenching of the
cocktail solutionmust be considered (i.e., dye molecules might absorb the fluorescence
signal from the fluor molecules in the cocktail and reduce the signal levels; chemicals
such as nitromethane might absorb the energy as heat and reduce the signal).
Imaging Instruments
To visualize the distribution of the radiotracer in vivo of an intact living organism
or tissue, radiation imaging techniques are used, such as autoradiography, Single Photon
Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET).
4
Chapter 1
Autoradiography normally uses a plate or film coated with special chemicals that
can react with ionizing radiation and subsequently indicate where the radiotracer is
distributed. The photographic film emulsion (silver halide) is the classical technique for
in vitro localization studies for planar objects. Digital radiography (reading the
photographic film with a digital scanner) has developed to not only locate the radioactive
sites (qualitative analysis), but also quantify the amount of activity using image analysis
software (e.g., Image Quant TL software). A phosphor imaging (PI) screen is similar to
the film used in autoradiography, but coated with photo-stimulable phosphor crystals,
barium fluorobromide containing a trace amount of bivalent europium as a luminescence
center (formulated by FujiFilm company as BaFBr: Eu2+) [1]. The crystal can trap and
store the energy of the ionizing radiation until scanned by a laser beam, which releases
the energy as luminescence. This phosphor technology was first used for medical X-ray
diagnoses and later found to be useful for other radioisotope detection in scientific
studies, such as
11
C,
14
C,
35
S,
59
Fe and
203
Pb. Besides X-rays, the PI screen can also be
used for β- and β+ detection.
SPECT cameras (gamma camera) are rotating scintillation detectors used to
acquire tomographic projection data, which are then reconstructed into 3D images.
SPECT is sensitive to samples with γ radiation. PET cameras are detector rings made of
scintillation crystals and photomultipliers to detect pairs of γ-rays generated by the
annihilation of positions (emitted from the radiotracer). SPECT and PET have improved
signal-to-noise ratio over autoradiography, and are able to provide quantitative
information for stationary or dynamic studies without damaging the sample. Both SPECT
5
Chapter 1
and PET are often combined with computed tomography (CT) or magnetic resonance
imaging (MRI) techniques to give both anatomic and distribution information.
1.3 Advantages and Disadvantages of Radiotracer Studies
One of the advantages of radiotracer studies is the high sensitivity of detection,
which far exceeds the detection limits of most other chemical or physical methods.
Because the detection is based on radiations from every atom, decent signals can be
achieved even with very low concentrations (pM or nM scale) of radiotracers present in
the system. This facilitates the study of metabolic substances that are normally present in
biological tissues at such low concentrations as to defy the most sensitive chemical
methods of identification (e.g., tracing the distribution of micronutrients in plants).
Another advantage of radiotracer studies is the possibility to coordinate the
dynamic processes within living systems with changes in metabolism. For example, both
metal uptake and distribution, and carbon metabolism and allocation in plants could only
be approached indirectly before the advent of radiotracer methods. Unfortunately, the fact
that radiotracer studies commonly involve such complex dynamic conditions often makes
it difficult to interpret the experimental results.
Despite the advantages mentioned above, radiotracers studies also have their own
issues that need to be addressed, such as radiation detection, radiation safety and, most
importantly, the experimental design.
1.4 Underlying Assumptions in Design of Radiotracer Experiments
1.4.1
Basic Assumptions
6
Chapter 1
Results of radiotracer experiments will be valid only if the following assumptions
are true.
1) There is no significant isotope effect, which means a radioactive isotope
behaves chemically and physically identical to the stable isotopes of the same element
[2]. It is not exactly true because the difference in masses between radiotracer nuclei and
stable nuclei (such as isotopes of hydrogen) can cause a shift in the reaction rate or
equilibrium (i.e., the isotope effect) [3]. However, in most cases (elements with a larger
mass number than 25), the isotope effect does not significantly affect the actual results.
The isotope effect of radioactive isotopes needs to be evaluated before the actual
radiotracer experiment.
2) There is no significant radiation damage to the experimental system. It is
essential that radiation from the radiotracer does not interfere with the normal biological
activities in the system of interest and subsequently distort the actual experimental
results. A minimum of radioactivity necessary for a reasonable counting rate is preferred
in the radiotracer experiments, especially for α- or β-- emitting tracers, to avoid damaging
the biological systems.
3) There is no deviation from the normal physiological state. In order to reach
the minimum activity required in the experiment for detection, the concentration of the
administered radiotracer could exceed the normal physiological levels of this compound
in the biological system and make the results open to question. Therefore, the specific
activity of the radiotracer should be high enough so that the concentration of the
radiotracer administered to the system can be within the normal range.
7
Chapter 1
4) The chemical form of the radioisotope-labeled compound is identical to the
unlabeled variety. Reactor production of radioisotopes often results in side reactions,
which can cause impurities (isotopic impurity or radiochemical impurity) in the
radiolabelled tracers administered to experimental systems. The impurities may interfere
with the actual radiotracer and affect the results. Therefore, it is important to take the
purity of the radiotracers into consideration.
5) Only the labeled atoms are traced. The radioactivity traced in the radiotracer
experiment is based on the labeled atoms, not necessarily the intact radiotracer compound
administered. The radiotracer compound could be involved in metabolic processes and
subsequently cleaved to form other intermediate metabolites.
1.4.2
Evaluation of the Feasibility of Radiotracer Experiments
It is critical to carefully design the radiotracer experiment and generate minimum
amounts of disposable radioactive waste. Several key factors need to be considered to
ensure the feasibility of a radiotracer experiment.
1) Availability of the radiotracer. A primary concern is if a radioisotope of the
element to be traced is available with the useful nuclear properties (i.e., half-life and
particle energy).
A second concern is if the radiotracer compound needed is
commercially available or can be easily obtained.
2) Limits of detection. The dose of administered radiotracer compound can be
diluted by the experimental system, or only a fraction of the administered compound is
taken up. Both situations affect the ability to detect and quantify the amount of the tracer
8
Chapter 1
present in the system. Thus, the amounts of administered radiotracer present at time
points after administration in the experimental system need to be evaluated prior to
determining the final protocol for the radiotracer experiments.
3) Evaluation of Hazard. The primary hazard concern is the possibility of harm
to the experimenter or co-workers. The hazard from direct external radiation can pose a
serious problem if proper shielding is not used. The physical form of the radiotracer (i.e.,
volatile liquid or solid powder) sometimes requires extra safeguards because it can be
potentially inhaled or ingested. The radioactive waste disposal protocol might require
modification if the radiotracer experiments are conducted in a biological system that
might make the waste bio-hazardous. Additionally, the non-radioactive tracer might be
toxic (e.g., Hg ions), which can make it more difficult to handle if the radioactive isotope
is incorporated for a radiotracer study.
1.5 History of Plant Biological Studies using Radioactive Isotopes
Using radiotracers to study plant systems stems back to 1923 when George de
Hevesey first developed radiotracer methods and later used radioactive Pb to study Pb
uptake and translocation in plants [4]. With the production of artificially produced
radioactive isotopes (11C,
13
N, 15O, 18F) [5-8] and the stable isotopes (15N,
13
C and
18
O),
the radiotracer method was gradually recognized and used in many research fields, such
as plant biology. In 1938, Martin Kamen and Sam Ruben were studying the carbon
metabolic pathway in photosynthesis by incorporating the short-lived radioisotope
into their experiments [9]. After Martin discovered another carbon isotope
cyclotron,
14
14
11
C
C using a
C was used as a radiotracer to study particular metabolic pathways in plant
9
Chapter 1
photosynthesis [10-13]. Kamen and Ruben used 14C to investigate “dark fixation” in plant
photosynthesis. Also, Melvin Calvin and his colleagues, using 14C, mapped the complete
route that carbon travels through a plant during photosynthesis, starting from its
absorption as atmospheric carbon dioxide to its conversion into carbohydrates and other
organic compounds [10]. Nowadays, with the increasing availability of radioisotopes,
commercially available radiolabeled compounds, and better radiation detection
techniques, radiotracer methods have become more useful to plant biologists for
investigating the basic mechanisms of nutrient metabolism and translocation in plants.
Radiotracer experiments are also being used as complimentary evidence to traditional
non-radioactive experiments.
1.6 Radiotracer Studies in This Dissertation
This work uses radiotracers to investigate basic nutrient metabolism and
translocation in plants using radiotracers in Arabidopsis thaliana.
Arabidopsis is a member of the mustard family, which includes cultivated species
such as cabbage and radish. Although Arabidopsis is not of major agronomic
significance, it offers important advantages for basic research in genetics and molecular
biology [14]. For example, Arabidopsis has a rapid growth cycle (6 weeks from
germination to reproduction), prolific seed production and is easy to cultivate. Also,
Arabidopsis has a small genome that has been sequenced, can be transformed easily by
agrobacterium, and a large number of mutant plants are commercially available. Such
features make Arabidopsis a good model for plant biological studies. Arabidopsis has
10
Chapter 1
different ecotypes. Columbia-0 (col-0) is used as a control (wild type, WT) in all the
studies mentioned in the dissertation.
Below is an outline of the radiotracer studies discussed in this dissertation and the
radiotracers used for the studies.
Chapter 2 describes a study of heavy metal accumulation in the opt3-2 mutant
(which was shown to over-accumulate Fe and several other di-valent cations in aerial
tissues) and the WT Arabidopsis.
203
Pb2+ was used to investigate uptake behavior and
distribution patterns in the mature opt3-2 mutant and WT plants.
203
Pb2+ was purchased
from Lantheus Medical Imaging and came dissolved in 0.5 M HNO3.
Chapter 3 describes a study of carbon metabolism and allocation in three starch
mutant plants, sex1-1 (starch excess), adg1-1 and pgm-1 (starch deficient), and WT
plants. 11CO2 was used to explore the short-term carbon flux in the starch mutant plants.
11
CO2 was produced by the cyclotron in the Brookhaven National Laboratory (BNL).
Chapter 4 describes a study of how carbon metabolism and allocation are
affected by the external and internal iron status in Arabidopsis mutants (opt3-2 and irt11) compared to the WT plants. In this study,
59
Fe3+ and
11
CO2 were both used to
investigate the dynamic relationship of carbon metabolism and Fe status within a short
time window.
11
59
Fe3+ was purchased from the Perkin Elmer and dissolved in 0.5 M HCl.
CO2 was produced by the same method mentioned in Chapter 3.
11
Chapter 1
References
1.
http://www.sb.fsu.edu/~xray/Manuals/ip.pdf.
2.
Choppin, G.R.,
http://oregonstate.edu/instruct/ch374/ch418518/Chapter%20IV%20Radiotracers.pdf.
Nuclear chemistry. 2nd edition.
3.
Lanigan, G.J., et al., Carbon isotope fractionation during photorespiration and
carboxylation in Senecio. Plant Physiol, 2008. 148(4): p. 2013-20.
4.
Hevesy, G., The absorption and translocation of lead by plants. A contribution to the
application of the method of radioactive indicators in the investigation of the change of
substance in plants. Biochemistry Journal, 1923. 17: p. 7.
5.
http://www.nasonline.org/publications/biographical-memoirs/memoir-pdfs/lauritsencharles.pdf.
6.
Ferrieri, R.A., et al., On-line production of 13N-nitrogen gas from a solid enriched 13Ctarget and its application to 13N-ammonia synthesis using microwave radiation. The
International Journal of Applied Radiation and Isotopes, 1983. 34(6): p. 897-900.
7.
Fowler, J.S., Wolf, A.P., Synthesis of carbon-11, fluorine-18, and nitrogen-13 labeled
radiotracers for biomedical applications, in Other Information: Portions of document are
illegible. 1981. p. Medium: ED; Size: Pages: 124.
8.
Ido, T., et al., Labeled 2-deoxy-D-glucose analogs. 18F-labeled 2-deoxy-2-fluoro-Dglucose, 2-deoxy-2-fluoro-D-mannose and 14C-2-deoxy-2-fluoro-D-glucose. Journal of
Labelled Compounds and Radiopharmaceuticals, 1978. 14(2): p. 175-183.
9.
Ruben, S., et al., Photosynthesis with Radio-Carbon. Science, 1939. 90(2346): p. 570571.
10.
Calvin, M., Benson, A. A., The Path of Carbon in Photosynthesis. Science, 1948.
107(2784): p. 476-480.
11.
Gest, H., Samuel Ruben's Contributions to Research on Photosynthesis and Bacterial
Metabolism with Radioactive Carbon. Photosynth Res, 2004. 80(1-3): p. 77-83.
12
Chapter 1
12.
Ruben, S., Kamen, M. D., Photosynthesis with Radioactive Carbon. IV. Molecular
Weight of the Intermediate Products and a Tentative Theory of Photosynthesis. Journal of
the American Chemical Society, 1940. 62(12): p. 3451-3455.
13.
Ruben, S., Kamen, M. D., Long-Lived Radioactive Carbon: C-14. Physical Review,
1941. 59(4): p. 349-354.
14.
http://www.arabidopsis.org/portals/education/aboutarabidopsis.jsp.
13
Chapter 2: Accumulation of Heavy Metals by the Arabidopsis opt3-2
Mutant
2.1 Isotope Summary
Isotopes Half-life
203
Pb
51.9 h
Decay Mode
E
EC (100%), γ
0.975 MeV (EC), 0.279 MeV (γ, 81%)
2.2 Background
Metals are naturally present in the Earth’s crust at various levels [1]. Mining,
industry, and agriculture lead to accelerated release of metals into ecosystems, causing
serious environmental problems as well as posing a threat to human health [2]. Although
many metals are essential for cells (e.g., Cu, Fe, Mn, Ni, Zn), all metals are toxic at
higher concentrations [3]. More than 50,000 metal-contaminated sites await remediation
in the U.S. alone. Approximately 80% of U.S. Superfund sites (designated by the U.S.
Environmental Protection Agency as priority sites for cleanup) contain heavy metals,
often mixed with organic pollutants [4].
Heavy metal contamination in the environment, if not carefully treated, can be
harmful to biological systems since heavy metals are not biodegradable and can
accumulate in living organisms such as plants, which are at the low end of the food chain.
If consumed as daily food, heavy metals could easily reach toxic levels in animals and
humans. Heavy metal contamination also poses dangers to the areas surrounding the
contaminated sites due to possible migration as dust, leachates through soil, or untreated
14
Chapter 2
sewage sludge. There are various methods to clean up heavy metal contaminated sites
and reduce the levels of toxicity, but most of them are costly, do not reach their optimum
performance, and also no living organisms can use the soil during the remediation
process.
Using a natural bio-process to clean up contaminated sites would be more ideal.
Phytoremediation is not a new concept [2, 5-10], but recently, has been revisited due to
its efficiency, affordable cost and most importantly environmental friendliness.
Phytoremediation involves using plants to clean up the contaminated environments by
removing, containing or transforming hazardous contaminants, such as heavy metals, to
improve environmental quality. Research has shown that certain plant species have
unique and selective uptake capabilities for heavy metals in their root systems and have
translocation, bioaccumulation, and detoxification abilities through the whole plant body
system [8, 11-15]. These features make plants a promising alternative for environmental
remediation.
Previous research has shown that Arabidopsis oligo-peptide transporter3-2 (opt32) mutant plants were able to over-accumulate Fe and some other di-valent cations, such
as Mn, Zn and Cu, due to the constitutively up-regulated expression of iron-regulated
transporter1-1 (IRT1-1) [16, 17]. IRT1-1 is an efficient iron transporter found in
Arabidopsis roots. IRT1 not only can transport Fe2+ into the plant root system, but also
can transport certain di-valent cations, such as Mn, Zn, Cd, etc. [18, 19]. All of the
features mentioned above suggest that IRT1-1 could play a role in transporting heavy
metals, and likely explains the over-accumulation of these metals in the tissues of the
opt3-2 mutant. This makes the opt3-2 Arabidopsis mutant a model system for studying
15
Chapter 2
the general mechanisms of heavy metal accumulation in plants, which could be later
translated to better plant systems (e.g., poplar trees) for phytoremediation purposes.
In this study, inductively-coupled plasma mass spectroscopy (ICP-MS) and
radioactive tracer (203Pb2+) assays were used to investigate heavy metal accumulation in
the tissues of the opt3-2 Arabidopsis mutant, as well as their uptake behavior and
distribution patterns. Transport assays using yeast were also used to further explore the
possible accumulation mechanism.
2.3 Materials and Methods
2.3.1
Materials
Bleach used for seed sterilization was purchased from Pure Bright (Columbia,
MO). Premier Pro-Mix soil was purchased from Quakertown, PA, USA. The restriction
enzyme HindIII was purchased from New England BioLabs (Ipswich, MA). DifcoTM
Yeast Nitrogen Base (without amino acids) was purchased from Becton, Dickinson and
Company (Franklin Lakes, NJ). Triton X-100, Murashige and Skoog (MS) salts with
vitamins, HgCl2, casamino acids, L-tryptophan, adenine and agar were purchased from
Sigma (St. Louis, MO). Sucrose, Pb(acetate)2 and MES (2-(N-morpholino)ethanesulfonic
acid, monohydrate) were purchased from Fisher Scientific (St. Louis, MO).
203
Pb(NO3)2 was purchased from Lantheus Medical Imaging (N. Billerica, MA).
2.3.2
Plant Growth Conditions
Seeds were surface-sterilized with a 35% bleach solution containing 0.1% Triton
X-100 for 20 min and then rinsed several times with sterile water. Prior to germination
seeds were placed in the cold room (4oC) for 2–3 days to align the seedling germination
16
Chapter 2
times. For routine growth, seedlings were grown in agar medium containing one-halfstrength Murashige and Skoog (MS) salts, MS vitamins, 1% sucrose (w/v), 0.05% MES.
The pH of the medium was adjusted to 5.7 with 5 M NaOH solution (conc?). For metal
uptake assays, one-week-old seedlings were transferred to the medium with different
concentrations of Pb(acetate)2 (500 µM, 750 µM) or HgCl2 (10 µM, 20 µM) . For root
analysis, petri dish plates were placed vertically. For seed amplification, two-week-old
seedlings were transferred and grown in Premier Pro-Mix soil. For ICP-MS analysis,
plants were grown under the same conditions as used for seed amplification except that
the soil was treated with Pb or Hg salts. For Pb treatment, 500 µM Pb(acetate)2 solution
was used to water the plants during the growth period. For Hg treatment, the soil was premixed with HgCl2 salts. Plants were all grown at 22°C under 100 µmole m-2 s1
fluorescent white light with an 8 hr (day)/16 hr (night) photoperiod. For 203Pb2+ uptake
assay, seeds were germinated in sand mix (QuikreteTM all-purpose sand and Sta-Green TM
Vermiculite; sand : vermiculite (v/v) was 5:1; sand was washed with tap water several
times, then washed with distilled water 4 times, then mixed with half-strength MS-soaked
vermiculite) and grown for 1.5 months. The growth conditions were the same as
mentioned above except the photoperiod was 12 hr (day)/12 hr (night) for this
experiment. This photoperiod resulted in plants that grew slower and with larger leaves,
which was preferred for the radiotracer experiments. The wild type (WT) Arabidopsis
used in this study was Columbia-0 (col-0).
17
Chapter 2
2.3.3
Determination of Chlorophyll Content
Plants were grown under the same conditions as mentioned above in
the plant growth assay. All the petri dish plates were placed horizontally and the
seedlings were grown for 2 weeks in the growth chamber. 5-8 seedlings were
selected from both wild type and opt3-2 mutant plants grown under different metal
treatments. Each seedling was put into a 1.5 ml Eppendorf tube and the chlorophyll was
extracted with 1 ml of 100% methanol. The extract solutions (200 µl) were pipetted into a
96-well plate and measured with a Bio-TEK plate reader at 652, 665 and 750 nm. The
chlorophyll content was calculated using the following equations [20] :
Chl a = [16.29 x (A665 – A750)] – [8.54 x (A652 – A750)]
Chl b = [30.66 x (A652 – A750)] – [13.58 x (A665 – A750)]
Chl a+b = [22.12 x (A652 – A750)] + [2.71 x (A665 – A750)]
A652, A655 and A750 indicate the absorbance at 652, 655 and 750 nm, respectively.
Chl a: Chlorophyll a; Chl b: Chlorophyll b; Chl a+b: Chlorophyll a and Chlorophyll b.
2.3.4
ICP-MS Analysis
The above-ground tissue of one-month-old pot-grown Arabidopsis plants were
harvested and separated into rosette leaves and influorescence stems, and then ground
into powders in liquid N2. ICP-MS analysis of Hg was done at the University of
Missouri Research Reactor (MURR). ICP-MS analysis of Pb was done at the
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National
Laboratory (PNNL)
18
Chapter 2
2.3.5
Radioactive 203Pb2+ Uptake Assay and Imaging
One-and-a-half month old plants grown in sand/vermiculite mixtures (s/v
mix) were used for the 203Pb uptake assay. Plants were removed from the pot by
immersion in water to loosen the s/v mix while keeping the roots intact. The plants were
washed gently with tap water and kept in a falcon tube filled with 0.5 mM CaCl2 solution
for 15 min and then transferred to another tube with half-strength MS medium and
equilibrated for 6 hours. The experimental set-up is shown in Figure 2-5A. Just before
adding 200 µCi of 203Pb(NO3)2, non-radioactive carrier Pb(acetate)2 was added into the
medium to reach a final concentration of 250 µM. The medium solution was stirred to
uniformly distribute the Pb2+ and the plants were incubated for 24 hours. The roots were
rinsed with 0.5 mM CaCl2 solution followed by distilled water to remove any surfaceadhered Pb2+. The plants were dried, weighed and cut into shoots and roots, then counted
using a NaI (Tl) well detector. Individual rosette leaves were cut off, displayed away
from the rosette leaf center and then exposed on the phosphor-image screen (FujiFilm),
which was later analyzed using a phosphor-imager (Typhoon 900).
2.3.6 Yeast Transformation and Growth Assays
The AtIRT1-1 gene was sub-cloned into the yeast expression vector pDB20
(pAtIRT1-1 was obtained from Stacey’s Lab). pAtIRT1-1 and vector pDB20 were
individually transformed into S. cerevicea yeast strain BY4730. The transformed yeast
was selected from minus-Uracil (-Ura) medium (means media without uracil). To
confirm the transformation, the two plasmids were extracted from BY4730 yeast,
amplified in E. coli and digested with restriction enzyme HindIII. Synthetic Defined
19
Chapter 2
medium without uracil (SD-Ura) was used, containing 0.67% Yeast Nitrogen base
(without amino acids), 0.1% casamino acids, 0.01% tryptophan and adenine. (protocols
were obtained from Stacey’s lab and attached in Appendix B). One colony of yeast was
inoculated in 3 ml of SD-Ura medium and shaken at 180 rpm at 30 ˚C overnight (final
OD600= 0.9-1.2). The cells were centrifuged at 4500 rpm for 6 min at 25 ˚C. After
decanting the supernatant, the cells were washed three times with sterilized ultrapure H2O. The cells were re-suspended in ultra-pure H2O and added to 3 ml of new SDUra medium to achieve an OD600= 0.03-0.05. Yeast was grown in the medium without
heavy metals for ~ 3 hr, after which time heavy metal solutions (HgCl2, Pb(acetate)2,
CdCl2) of different concentrations were added. The growth of cultures was scored over
the period of 20 or 24 hours by measuring the OD600.
2.3.7 Statistical Analysis
Data was subjected to the Student t-test for unpaired samples assuming an
unequal variance. Statistical significance levels were assigned to the following rating
scale (*, p<0.05; **, p<0.01; ***, p<0.001).
2.4 Results
2.4.1
Toxic Effects of Heavy Metals on Plant Growth
Heavy metals are known to be toxic to plants and can significantly inhibit plant
growth and physiology, such as shoot/root growth and chlorophyll synthesis [14]. To
investigate the response of the opt3-2 mutant relative to the WT, both were grown in
media with different concentrations of Pb2+ or Hg2+, and several physiological properties
were measured. (Fig. 2-1 and Fig. 2-2) Shoot and root growth of both the opt3-2 mutant
20
Chapter 2
and the WT plants were inhibited when heavy metal ions were introduced into the
medium, especially at the higher concentration of Pb2+ or Hg2+. The shoot growth of the
opt3-2 mutant was less than the WT even in the absence of heavy metals. However, in the
absence of heavy metals, the chlorophyll content of the mutant and the WT plants were
identical. This was not the case when heavy metals were added; the opt3-2 mutant plants
showed significantly lower chlorophyll content, which was also observed visually when
seedlings were grown in the agar plates. (Fig. 2-1) The root growth of the opt3-2 mutant
was similar to the wild type under the control conditions, but was significantly inhibited
in the presence of high concentrations of heavy metal ions. Compared to the wild type,
the opt3-2 mutant had less root mass, especially fewer lateral roots. (Fig. 2-2)
2.4.2
Heavy Metal Quantification in Plant Tissues by ICP-MS
To quantify the amount of heavy metal taken up and stored in the aerial plant
tissues, influorescence stems (including leaves, flowers and seliques) and rosette leaves
of both opt3-2 mutant and WT plants were ground into powders under liquid nitrogen and
analyzed by ICP-MS. Under control conditions, no heavy metals were added to the soil.
Therefore, the amount of Pb or Hg present in the plant tissues under the control
conditions is due to the natural amount of Pb or Hg present in the soil or water. Overall,
under Pb or Hg treatment, there was more Pb2+ or Hg2+ taken up in rosette leaves than
stems in both WT and opt3-2 mutant plants. The opt3-2 mutant showed significantly
more Pb2+ in both influorescence and rosette leaves compared to the wild type, which
indicates that opt3-2 over-accumulates Pb2+ in the above-ground tissues. (Fig. 2-3a) The
Hg data showed the same general trend as that seen with the Pb treatment. However, the
21
Chapter 2
amount of accumulation of Hg was ten thousand times lower than Pb and the differences
seen were not statistically significant.
Heavy metal uptake in the roots was investigated using radiotracers, not by ICPMS. (Fig. 2-6)
2.4.3
Radioactive 203Pb2+ Uptake Assay
Heavy metal uptake in the roots was investigated using radiotracers (Fig. 2-6).
The results from the 203Pb2+ uptake assays showed no significant difference between
opt3-2 and WT plants in the total amount of Pb taken up into the entire plant. There was
no apparent over-accumulation of Pb in the shoots of the opt3-2 mutant plants compared
to the WT. Both the opt3-2 mutant and WT plants stored over 90 percent of the Pb in the
roots and only moved a small fraction to the shoots (Fig. 2-6). As shown in the phosphor
image (Fig. 2-5), the Pb in the leaves was intensively localized in small spots, which are
possibly the trichomes or the stomata[7]. Further studies are needed to clarify this distinct
phenotype.
2.4.4
IRT1 Does Not Mediate Pb or Hg Uptake
To investigate the ability of IRT1-1 to transport Pb2+ or Hg2+, BY4730 yeast cells
was transformed with the pAtIRT1-1. Yeast cells with transformed pDB20 (an empty
vector) were used as a negative control. Pb2+/Hg2+ is toxic to yeast and, therefore, if
IRT1-1 does transport these metals, we would assume that yeast expressing this
transporter would accumulate more Pb2+/Hg2+ and their growth would be inhibited
significantly more than cells expressing the empty vector.
22
Chapter 2
Yeast cells were first grown in a medium with Cd2+, a known substrate for IRT1,
to test if AtIRT1-1 was expressed and functioning correctly [21]. As shown in Figure 24b, the growth of yeast expressing AtIRT1-1 was inhibited significantly when Cd2+ was
added to the medium compared to yeast transformed with pDB20, while in Figure 2-4a
both transformed yeast cells grow similarly with normal nutrient medium. These results
indicate that the yeast cells are expressing pAtIRT1-1, which is mediating the uptake of
Cd. However, in contrast to the results obtained with Cd, there was no apparent
difference in growth for the yeast strains under different concentrations of Pb2+ or Hg2+
(Fig. 2-4). Both transformed yeast lines were affected to a similar level with or without
pAtIRT1-1. In addition, both yeast transformants exhibited a better tolerance to Pb than to
Hg. Compared to the Hg treatment, both yeast transformants grew much better even in
the presence of the higher concentrations of Pb.
2.5 Discussion
Our current results show that compared to wild type Arabidopsis, the opt3-2
mutant over-accumulates Pb and Hg in the aerial tissues (Fig. 2-3). This makes the
opt3-2 mutant a good system for investigation of the underlying mechanism of heavy
metal accumulation in plants, which is very important for developing and improving
engineered plants for phytoremediation purposes.
The root growth of opt3-2 mutant plants were more affected by heavy metal
treatment than the wild type, although both the opt3-2 mutant and the wild type were
inhibited when high concentrations of Pb2+/Hg2+ were present. Several studies have
shown that Pb2+ and Hg2+ are primarily accumulated in plant roots relative to aerial
23
Chapter 2
tissues and can inhibit root growth (including lateral root growth) dramatically[11, 14,
22]. As seen in Figure 2-2, both the main root and lateral root growth were inhibited
when seedlings were grown under high concentrations of Pb2+/Hg2+, especially opt3-2
mutant plants.
In general, under high concentrations of heavy metals, the shoot growth of both
the opt3-2 and wild type was inhibited in terms of shoot mass and chlorophyll content
(Fig. 2-1). Hg can be transported into the leaves and it is known to affect photosynthesis
and oxidative metabolism by interfering with electron transport in chloroplasts and
mitochondria [23, 24]. Pb may also directly or indirectly affect photosynthesis. As
shown in Figure 2-1, the opt3-2 mutant showed significantly lower chlorophyll content
under heavy metal treatment, which may be due to a substitution for Fe by the heavy
metal to affect the normal chlorophyll synthesis [25, 26]. The opt3-2 mutant also showed
a consistently lower shoot mass than the WT, even in the absence of heavy metal
addition. This may be due to the over-accumulation of other metals, such as Fe, which
occurs in this mutant even when excess Pb or Hg is not present [16].
In the aerial portion of the plants, as shown in Figure 2-3, the major accumulation
was observed in rosette leaves for both Pb and Hg. There was much less Pb/Hg present in
the stems. This indicates that rosette leaves are sinks for Pb2+/Hg2+ once they are taken
up, immobilized and stored in certain regions of the cell, such as the cell wall and the
vacuoles [7, 11, 24].
The 203Pb uptake assay showed there was no significant difference in Pb
accumulation between the opt3-2 and the wild type within a 24-hour window. (Fig. 2-6)
24
Chapter 2
These results are different from the data seen using ICP-MS analysis. The results suggest
that the initial rate of metal uptake (measured by 203Pb uptake) is not affected in the opt32 mutant plants but long term accumulation of heavy metals (as measured by ICP-MS
analysis) is strongly affected in the mutant. These data are consistent with the notion that
OPT3 does not mediate metal uptake in the roots but rather is involved in internal
mobilization of metals within the plant [16]. Most of the studies on heavy metal
accumulation only show the cumulative amount of metals in the plant tissues. However,
using radiotracer assays to explore what is happening during the accumulation process
allowed us to distinguish between short term uptake versus long term accumulation
mechanisms.
Hg, unlike Pb, was not shown to be over-accumulated in any of the tissues of the
opt3-2 mutant according to the ICP-MS results (Fig. 2-3). However, it is possible that
some of the Hg that was taken up by the plants was transformed into Hg0 through plant
biological processes; Hg0 is volatile and easily released into air. This process may result
in a loss of Hg content in both of the WT and the opt3-2 mutant, which could affect the
accuracy of ICP-MS analysis.
In the past, the over-accumulation of Fe and Cd in opt3-2 mutant plants has been
explained by the constitutive, up-regulation of the IRT1 transporter in this mutant [16].
Indeed, IRT1-1, although initially described as an iron transporter, does transport Cd, as
well as a few other divalent cations. However, IRT1 is not known to transport either Pb
or Hg. In order to test this directly, we expressed AtIRT1 in yeast and measured the
effects of heavy metal addition on yeast growth. In control experiments, yeast expressing
AtIRT1 showed increased sensitivity to Cd addition, consistent with the expression of a
25
Chapter 2
functional transporter. However, yeast expressing AtIRT1 showed no increased sensitivity
to the toxic effects of Pb or Hg. Therefore, we conclude that up-regulation of AtIRT1 in
opt3-2 mutant plants is unlikely to be the explanation for the increased sensitivity and
accumulation of Pb or Hg by these plants. The analysis of DNA microarray data
(unpublished data from David G. Mendoza-Cozatl, data not shown) comparing the opt3-2
mutant to wild type indicates that the expression of a number of transporter genes is
affected in the mutant, in addition to AtIRT1. It is possible that one or more of them may
be responsible for Pb/Hg trafficking.
26
Chapter 2
Figure 2-1. Shoot growth in different heavy metal treatments. upper panel: a, without heavy
metal; b and c, with 500µM and 750µM Pb(acetate)2; d and e, with 10µM and 20µM HgCl2.
col-0 is on the left side while opt3-2 is on the right side. Seedlings are two weeks old. Lower
panel: shoot mass (n=12 seedlings) and chlorophyll content (n=8 seedlings, mean ± SE) under
different heavy metal treatments. col-0 is the wild type Arabidopsis used in all the studies. See
Table A1-3 and A1-4 in Appendix A for tabular data.
27
Chapter 2
Figure 2-2. Root growth in different heavy metal treatments. Left panel: a, without heavy metal;
b and c, with 500µM and 750µM Pb(acetate)2; d and e, with 10µM and 20µM HgCl2. col-0 is on
the left side while opt3-2 is on the right side. Seedlings are two weeks old. Right panel: Root
mass (n=12 seedlings, mean ± SE) under different heavy metal treatments. See Table A1-3 in
Appendix A for tabular data.
28
Chapter 2
Figure 2-3. ICP-MS shows the amount of heavy metals in different above-ground tissues of both
col-0 and opt3-2 mutant. Left: Pb content; Right: Hg content. Plants used in this experiment are
one month old. n=2-3, mean ± SE. See Table A1-5 and A1-6 in appendix A for the tabular data.
29
Chapter 2
a.
b.
c.
d.
e.
f.
Figure 2-4. Comparison of different yeast transformants grown under various concentrations of
heavy metal treatments. a) control, b) 5µM Cd2+, c) 125 µM Pb2+, d) 250 µM Pb2+, e) 5 µM Hg2+
and f) 8 µM Hg2+. n=3, mean ± SE
30
Chapter 2
Figure 2-5. Phosphor image of 203Pb distribution in opt3-2 mutant and wild type plants. A, set-up
for Pb-203 uptake assay; B, Left one is wild type (col-0) and right one is opt3-2 mutant. Arrows
are pointing out the leaves with concentrated spots which are very likely Pb accumulation in
trichomes.
a.
b.
Figure 2-6. 203Pb distribution in opt3-2 mutant and wild type plants. a, total counts of Pb
in the entire plant tissues (roots and shoots); b, counts of Pb in roots and shoots
separately. n=4, mean ± SE. See Table A1-7 in AppendixA for the tabular data.
31
Chapter 2
Table 2-1. Fractions of total 203Pb taken up in the plant transported into roots and shoots of the
WT (col-0) and the opt3-2 mutant
Type of plant
% of total 203Pb
% of total 203Pb
Transported to the roots
Transported to the shoots
col-0
99.80 ± 0.03
0.20 ± 0.03
opt3-2
99.84 ± 0.07
0.16 ± 0.07
32
Chapter 2
References
1.
Angelone, M., Bini, C., Trace element concentrations in soils and plants of Western
Europe. In: Biogeochemistry of Trace Metals, 1992: p. 19-60.
2.
Pilon-Smits, E., Pilon, M., Phytoremediation of Metals Using Transgenic Plants. Critical
Reviews in Plant Sciences, 2002. 21(5): p. 439-456.
3.
Marschner, H., Mineral Nutrition of Higher Plants Academic. Press, London, 1995.
4.
Ensley, B.D., Rationale for use of phytoremediation. In: Phytoremediation of Toxic
Metals — Using Plants to Clean up the Environment. Raskin, I. and Ensley, B.D., Eds.,
Wiley, New York, 2000: p. 1-12.
5.
Salt, D.E., Blaylock, Michael, Kumar, Nanda P.B.A., Dushenkov, Viatcheslav, Ensley,
Burt D., Chet, Ilan, Raskin, Ilya, Phytoremediation: A Novel Strategy for the Removal of
Toxic Metals from the Environment Using Plants. Nat Biotech, 1995. 13(5): p. 468-475.
6.
Hinchman, R.R., Negri, M. Cristina, Gatliff, Edward G. , Phytoremediation: Using
Green Plants to Clean up Contaminated Soil, Groundwater, and Wastewater. Argonne
National Laboratory Hinchman, Applied Natural Sciences, Inc, 1998.
7.
Clemens, S., Palmgren, M. G., Kramer, U., A long way ahead: understanding and
engineering plant metal accumulation. Trends Plant Sci, 2002. 7(7): p. 309-315.
8.
Ghosh, M., Singh, S. P., A review on phytoremediation of heavy metals and utilization of
its byproducts. Asian Journal on Energy and Environment, 2005. 6(4): p. 214-231.
9.
Krämer, U., Cadmium for all meals – plants with an unusual appetite. New Phytologist,
2000. 145(1): p. 1-3.
10.
Lasat, M.M., Phytoextraction of toxic metals: a review of biological mechanisms. J
Environ Qual, 2002. 31(1): p. 109-120.
11.
Liu, D., et al., Uptake and accumulation of lead by roots, hypocotyls and shoots of Indian
mustard [Brassica juncea (L.)]. Bioresource Technology, 2000. 71(3): p. 273-277.
12.
Prasad, M.N.V., Metal hyperaccumulation in plants-biodiversity prospecting for
phytoremediation technology. Electronic Journal of Biotechnology, 2003. 6(3): p. 285320.
33
Chapter 2
13.
Xiao, S., Chye, M. L., Arabidopsis ACBP1 overexpressors are Pb(II)-tolerant and
accumulate Pb(II). Plant Signal Behav, 2008. 3(9): p. 693-694.
14.
Tangahu, B.V., et. al., A Review on Heavy Metals (As, Pb, and Hg) Uptake by Plants
through Phytoremediation. International Journal of Chemical Engineering, 2011. 2011: p.
31.
15.
Lee, K.K., et. al., Cadmium and Lead Uptake Capacity of Energy Crops and Distribution
of Metals within the Plant Structures. Journal of Civil Engineering, 2013. 17(1): p. 44-50.
16.
Stacey, M.G., et al., The Arabidopsis AtOPT3 protein functions in metal homeostasis and
movement of iron to developing seeds. Plant Physiol, 2008. 146(2): p. 589-601.
17.
Garcia, M.J., et al., Shoot to root communication is necessary to control the expression of
iron-acquisition genes in Strategy I plants. Planta, 2013. 237(1): p. 65-75.
18.
Korshunova, Y.O., et al., The IRT1 protein from Arabidopsis thaliana is a metal
transporter with a broad substrate range. Plant Mol Biol, 1999. 40(1): p. 37-44.
19.
Vert, G., et al., IRT1, an Arabidopsis transporter essential for iron uptake from the soil
and for plant growth. Plant Cell, 2002. 14(6): p. 1223-1233.
20.
Porra, R.J., Thompson W.A., Kreidemann, P.E., Determination of Accurate Extinction
Coefficients and Simultaneous Equations for Assaying Chlorophylls a and b Extracted
with Four Different Solvents: Verification of the Concentration of Chlorophyll Standards
by Atomic Absorption Spectroscopy. Biochimica et Biophysica Acta, 1989. 975: p. 348389.
21.
Rogers, E.E., D.J. Eide, and M.L. Guerinot, Altered selectivity in an Arabidopsis metal
transporter. Proc Natl Acad Sci U S A, 2000. 97(22): p. 12356-60.
22.
Sharma, P., Dubey, R.S, Lead Toxicity in Plants. Braz. J. Plant Physiol., 2005. 17(1): p.
35-52.
23.
Morenoa, F.N., Andersonb, C.W.N, Stewartb, R.B., Robinson, B.H., Phytofiltration of
mercury-contaminated water: Volatilisation and plant-accumulation aspects.
Environmental and Experimental Botany, 2008. 62: p. 78-85.
24.
Ferreira, C., Lopes Vieira, C., Azevedo, H., Caldeira, G., The effects of high levels of Hg
on senescence, proline accumulation and stress enzymes activities of maize plants.
Agrochimica, 1998. 42(5): p. 209-218.
34
Chapter 2
25.
Spiller, S.C., Castelfranco, A. M., Castelfranco, P. A., Effects of Iron and Oxygen on
Chlorophyll Biosynthesis: I. In Vivo Observations on Iron and Oxygen-Deficient Plants.
Plant Physiology, 1982. 69(1): p. 107-111.
26.
Pushnik, J.C., Miller, G.W., Iron regulation of chloroplast photosynthetic function:
mediation of PSI development. J. Plant Nutr., 1989. 12: p. 407-421.
35
Chapter 3: Dynamics of Carbon Metabolism and Allocation in
Arabidopsis Starch Mutants Using 11CO2
3.1 Isotope Summary
Isotopes
11
C
Half-life
Decay Mode
Emax
β+ (99.8%),
0.96 MeV (β+),
EC (0.2%)
0.511 MeV annihilation photons (199.6%)
20.3 min
3.2 Background
A fundamental understanding of how higher plants allocate and utilize their
carbon-based resources for growth and development can be an important factor guiding
future research toward attaining sustainability of both agricultural and bioenergy
cropping systems.
Plants assimilate the fixed carbon into fructose and glucose during the day, which
is then used for sucrose synthesis to support daily biological activities and plant growth,
with the remaining sugars stored as transient starch that can be degraded at night back
into soluble sugars to be used as a source of energy.
Sucrose is the primary sugar in plants and can be loaded into the phloem of source
tissues (such as mature leaves) for long-distance transport to sink tissues (such as roots,
young leaves and flowers). Other forms of sugars (such as fumarate, sorbitol, mannitol
and raffinose) are used for long-distance transportation, but they are not preferred in
Arabidopsis plants [1-3]. Fructose and glucose are building blocks for sucrose synthesis,
36
Chapter 3
as shown in Equation 1 below. This reaction is reversible. Sucrose can be degraded into
fructose and glucose using enzymes, such as invertase or sucrose synthase.
Suc. synthase
Fructose 6-phophate + UDP-glucose
Sucrose
Invertase or
Suc. synthase
UDP: Uridine diphosphate
Equation 3-1: Sucrose synthesis and degradation.
Starch is the major storage carbohydrate in plants and there are two types of
starch, amylose and amylopectin. Amylopectin, a highly branched glucan made of α-1,4linked glucose units and branched at the α-1,6-bonds, makes up 70% to 80% of the
starch. The remaining 20% to 30% of the stored starch is in the form of amylose, a linear
glucan containing few α-1,6-branches. [4, 5] Plants assimilate and store a large portion of
fixed carbon as transient starch in the chloroplast during the day, and degrade it into
glucans at night to provide a steady supply of carbon as an energy source to support the
normal growth of plants. [6-8] Photosynthetic partitioning of carbon into starch is well
regulated. An imbalance in starch synthesis and degradation leads to changes in the pool
size of key carbon metabolites, affecting the plant’s overall growth and development. [4,
6, 9-14]
There are several important enzymes that play essential roles in starch synthesis
and degradation (Fig. 3-1). Adenosine diphosphate (ADP)-glucose pyrophosphorylase
(AGP) is considered the key enzyme for converting glucose-1-phosphate (glc-1P) into
ADP-glucose (ADPG), which is then used for subsequent starch biosynthesis [15].
Plastidic phosphoglucomutase (PGM) is another important enzyme used in starch
synthesis. PGM converts glucose 6-phosphate (glc-6P) to glc-1P, which is a precursor of
37
Chapter 3
ADPG (Equation 2) [16] and is found in both the plastids and the cytosol [17]. Starch
excess1 protein (SEX1), reported by Yu[18], is a protein with significant homology to the
starch granule-bound protein (R1) found in potato and plays an important role in
regulating starch mobilization by controlling the phosphate content of starch.
ATP + glucose 1-phosphate
AGP
diphosphate + ADP-glucose
ATP
glucose 6-phosphate
PGM
glucose 1-phosphate
Equation 3-2: Biological functions of the enzyme ADP-glucose
pyrophosphorylase (AGP) and phosphoglucomutase (PGM).
Starch metabolism has been extensively studied in the past [4-6, 8, 19], leading to
sound knowledge of the pathways and the enzymes involved. However, there are still
many open questions regarding the signals and mechanisms regulating starch
metabolism. To further explore this topic, several Arabidopsis starch mutants were used
to target specific steps in the starch metabolism pathway as well as to see how plants
respond physiologically to diurnal (time-of-day) effects.
Starch excess mutants
Starch excess Arabidopsis mutants exhibit over-accumulation of starch in leaf
tissues due to an impairment of starch degradation at night. The sex1 and the sex4
mutants have been studied in the past [9, 18, 20]. The sex1 mutant was shown, in
Trethewey and Rees’ study [21], to be incapable of exporting glucose produced by starch
hydrolysis in the chloroplast. However, in Yu’s study years later [18], the sex1 mutant
38
Chapter 3
was shown to lack the SEX1 protein instead of a hexose transporter. The sex4 mutant is
deficient in a chloroplastic endoamylase, which reduces the rate of starch degradation
[20]. Both starch excess mutants were found to contain a large amount of starch content
in the early morning compared to the wild type Arabidopsis (Columbia-0), and this high
starch level phenotype arises from the gradual accretion of starch during development.
Also, an altered carbon partitioning to soluble sugars was observed in the sex4 mutant
[9].
Starch deficient mutants
In general, starch deficient mutants exhibit little to no starch synthesized in
tissues. The adg1 and the pgm-1 mutants have been well studied in the past. The adg1-1
mutant is a monogenic recessive mutation with less than 2% of the starch of the wild
type. Bahaji’s research [15] showed that the adg1-1 mutant has much less ADGase
activity in leaf extracts, 2% of the ADGase activity of the wild type, but has comparable
amounts of ADP-glucose. Thus, there might be an alternate pathway to synthesize ADPglucose in the adg1-1 mutant and subsequently to synthesize starch. The pgm-1 mutant
lacks the plastidic PGM enzyme activity compared to the wild type and has very low
levels of starch synthesis. Since the assimilated carbon cannot be used to synthesize
starch, a large amount of the carbohydrates are stored in the form of soluble sugars.
Research has shown that the pgm-1 mutant has an increased pool size of sucrose and
hexose sugars [16, 22].
The plant carbohydrate metabolism is not only dependent on the internal carbon
status, but also can be affected by hormone levels, such as the indole-3-acetic acid
39
Chapter 3
(IAA/Auxin) level in plants. [5, 23] IAA is a plant hormone that plays a role in many
diverse developmental and cellular processes impacting axis formation and patterning
[24, 25], especially in regulating root development [26]. Most particularly, this hormone
is known to cause extensive lateral root patterning as well as extensive root hair
formation at the expense of root elongation. A recent maize study [23] showed that by
using the 11CO2 labeling technique, in combination with a continuous root treatment with
IAA, an alteration of new carbon partitioning into soluble sugars was affected along with
starch content. For example, an increasing amount of 11C-partitioning into sucrose rather
than glucose and fructose sugars was observed in the IAA treated plants.
In this study, we leverage a combination of molecular tools (12C and 11C analyses)
through the use of starch mutants (sex1-1, adg1-1, pgm-1) and the plant’s natural
evolutionary trait of diurnal cycling of carbon resources to explore the effects of these
parameters on the terminal phenotype.
Many studies have been done to investigate these starch mutants individually, but
a detailed comparison has not yet been conducted. In addition, previous carbon
metabolism studies have focused on 12C analysis, which demonstrates the total amount of
carbon in different forms in the plant tissues, but is limited in its use for detecting carbon
flux in a short time window. Carbon-14 (14C) chase assays have been widely used to track
the dynamics of carbon partitioning, however, 14C is a weak β- emitter with a long halflife requiring a long exposure time to acquire the amount of signal needed for analysis. In
our study, 11CO2 was used to explore the dynamics of carbon metabolic partitioning and
allocation during the daytime. Carbon-11 (11C) decays by positron emission with a short
half-life of 20.36 min. The positrons can be detected by a phosphor screen with high
40
Chapter 3
sensitivity and it gives excellent signal resolution with a relatively short exposure time,
even though the concentration of 11C is very low in plant tissues. Thus, 11C analysis is a
more efficient technique to study the quickly changing concentration of carbon in plant
tissues. All of the 11C studies were conducted at Brookhaven National Laboratory (BNL),
which has a unique facility capable of producing and administering discrete doses of
radioactive 11CO2 to intact plants.
3.3 Materials and Methods
3.3.1 Materials
Bleach used for seed sterilization was purchased from Pure Bright (Columbia,
MO). All other chemicals used in this study were purchased from Sigma Aldrich (St.
Louis, MO).
3.3.2 Plant Growth Conditions
Seeds used in this study were purchased from The Arabidopsis Information
Resource (TAIR).
Seeds were surface-sterilized with a 35% bleach solution containing 0.1% Triton
X-100 for 15 min, and rinsed several times with sterile water. Prior to germination seeds
were placed in the cold (4oC) for 2–3 days to align seedling germination times. For
routine growth, seedlings were grown in an agar medium containing fullstrength Hoagland’s salts, 0.05% morpholinoethanesulfonic acid (MES) and 5%
agar. The pH of the medium was adjusted to 6.0 with NaOH solution. Seedlings were
41
Chapter 3
grown at 22°C under fluorescent white light (100 µmole m-2 s-1) with a 12 h (day)/12 h
(dark) photoperiod.
3.3.3 Starch Staining Assay
Either 48–well or 96-well plates were used for this assay. Two-week old seedlings
were immersed in methanol and kept at 0
for 0 min before decanting all of the
extraction solution. This step was repeated another time to ensure complete chlorophyll
removal from the leaves. An aliquot of iodine solution (containing 0.34% I2 and 0.68%
KI) was then added to each well of the plate to cover the whole seedling. The seedlings
were stained for 30 min before being rinsed with deionized water. An upright microscope
(Olympus Vanox AHBT3) with a color digital camera (provided by the MU Molecular
Cytology Research Core Facility) was used for taking images. Leaf starch staining was
done both in the early morning (1 hour after the light turned on) and late afternoon (7
hours after the morning assay). Root tips were cut off from the root just before imaging
them.
3.3.4 11CO2 Production, Pulsing and Incubation Apparati
11
CO2 was produced via the 14N(p, α)11C nuclear transformation [27] from a 20
mL target filled with high-purity nitrogen gas (400 mL @ STP) using 18 MeV protons
from the TR-19 (Ebco Industries Ltd, Richmond, BC, Canada) cyclotron at BNL, and
captured on molecular sieves (4Å). The captured 11CO2 was desorbed and quickly
released into an air stream at 200 mL/min as a discrete pulse for labeling the seedlings in
customized sealed incubation chambers (Fig. 3-2). Seedlings within the chamber were
42
Chapter 3
pulse-fed 11CO2 for 1 minute, and then chased with normal air for the duration of the
exposure.
Two types of customized apparati were used as sealed systems during 11CO2
pulsing and incubation of the seedlings. For sugar analysis, a square petri dish (without
its cover) with seedlings grown in agar media was kept in a sealed tip box (Fig. 3-2A).
For the carbon allocation assay, 1.5 ml Eppendorf™ tubes with seedlings grown in agar
media were placed in a sealed customized tube rack (Fig. 3-2B). Both apparati have two
connections, one for 11CO2 introduction and one to serve as an exhaust for the closed
system. Fixed red/blue LED lights were placed on top of the tip box/ tube rack to ensure
normal photosynthesis at 100 µmole m-2 sec-1. Light intensity was monitored before each
experiment using a LiCor Quantum sensor.
The 11CO2 experiments were conducted both in the early morning (1 hour into the
start of the photoperiod) and late afternoon (7 hours after the AM experiment) as
described in the Starch Staining Assay.
3.3.5 Tissue Extraction and Soluble Sugars Analysis
Seedlings for sugar analysis (wild type and three starch mutants) were grown on a
square plate filled with half-strength MS agar medium for 2-3 weeks. After 11CO2 pulsing
and incubation for 20 min, all the leaf tissues were harvested and immediately frozen in
liquid nitrogen and ground by hand in a pre-weighed and pre-chilled Eppendorf™ tube.
Methanol, four-times the milligram mass of the powder, was added to the centrifuge tube,
vortexed (VWR analog vortex mixer; Sigma-Aldrich Corp., St. Louis, MO, USA) and
then sonicated (Branson Bransonic 32; Sigma-Aldrich Corp. St. Louis, MO, USA) at 0 C
43
Chapter 3
for 10 min. Additional vortexing was performed during this period to ensure complete
mixing. Afterward the Eppendorf tubes were centrifuged for 2 min at 15,000 rpm to
separate the insoluble portion from the soluble one. The insoluble portion contained
mostly cell-wall polymers and starch. The soluble portion contained small soluble
compounds, including soluble sugars. These sugars were separated and analyzed by thin
layer chromatography (TLC) [28, 29]. Glass backed NH2-silica HPTLC-plates (200 µm,
w/UV254) were used for the sugar separation (Sorbent Technologies, Atlanta, GA,
USA). Plates were pre-spotted with 2 µL of the sugar standards (including 4, 3, 2, 1, 0.5
and 0.2 mM of glucose, sucrose and fructose) using a semi-automatic Linomat 5 sample
applicator (Camag Scientific Inc., Wilmington, NC, USA) for a high precision of spot
size and sample volume. Aliquots (2 µl) of leaf extract were also spotted. TLC plates
were developed using a mobile phase consisting of 75:25 acetonitrile/water (v/v). The
developed TLC plates were imaged using a Phosphor screen (FujiFilm BAS-MS 2040,
GE Healthcare Biosciences, PA) to determine the yield of each radiolabeled sugar. The
plates were then heat-treated ( 00 C, 10 min) to initiate a chemical reaction of individual
sugars with the amino functionalized Si-support giving a fluorescence marker detectable
under long wavelength (365 nm) UV light [28]. Digital photographs were taken of the
fluorescent markers to provide a measure of the 12C-sugar yields. Image Quant TL
software was used to analyze both the radiographic and the digital images to determine
the amount of carbon-11 (11C) or carbon-12 (12C) sugars. Linear Log regression was
used for plotting standard curves generated from the 12C-sugar standards.
44
Chapter 3
3.3.6 Carbon Allocation
Seedlings for carbon allocation analysis were grown for 2-3 weeks in 1.5 ml
Eppendorf™ tubes filled with agar media. After 11CO2 pulsing and 1 hour incubation, the
entire seedlings were removed from the agar media and cut into roots and shoots, then
stored in respective vials filled with methanol. Tissues were later counted in a NaI(Tl)
well-counter. The remaining tubes, containing agar medium, were also placed
individually into empty vials for counting, which indicated the amount of 11C-labeled
root exudates. This experiment was conducted both in the early morning and late
afternoon as described in the 11CO2 production, pulsing and incubation apparatus.
The percentage of 11C-phytosynthates transported to below-ground and exuded
from the roots was calculated as follows:
% of 11C-phytosynthates below =
A roots + A exudates
× 100%
A shoots + A roots + A exudates
% of 11C-phytosynthates exudates =
A exudates
× 100%
A shoots + A roots + A exudates
A shoots, A roots and A exudates indicate the activities measured in the shoots, root tissues and
in the gel (root exudates).
45
Chapter 3
3.3.7 Phosphor Imaging
After the Carbon Allocation assay, some of the seedlings were removed from the 1.5 ml
Eppendorf™ tubes and exposed to a phosphor imaging screen (FujiFilm, BAS-MS grade)
for 20 seconds, which was then analyzed using a phosphor-imager (TyphoonTM FLA
9000, GE Healthcare).
3.3.8 Root Morphology
Seedlings were prepared using the same method as described in the Tissue
Extraction and Soluble Sugar Analysis. The area of the entire roots and lateral roots was
measured by RootSnap software (CID Bio-Science, Inc.).
3.3.9 Root Elongation and Gravitropic Response
Seedlings were germinated vertically in full-strength Hoagland’s agar medium for
5 days before transferring into fresh agar medium under the same conditions. eedlings
were placed perpendicular to the ground for a day as time zero. Then the plates
containing the seedlings were rotated 90 . ictures were taken over the first 24 hours.
Root lengths and angles were measured later by ImageJ software.
3.3.10 Statistical Analysis
Data was subjected to the Student t-test for unpaired samples assuming an
unequal variance. Statistical significance levels were assigned to the following rating
scale (*, p<0.05; **, p<0.01; ***, p<0.001).
46
Chapter 3
3.4 Results
3.4.1 Plant Growth
Compared to the wild type (WT, col-0), both starch excess and starch deficient
mutants showed stunted root growth (Fig. 3-3). Their roots grew slower than WT roots
(Fig. 3-4). Among the starch mutants, adg1-1 and pgm-1 mutants have significantly more
total root area developed than the sex1-1 and the WT plants, especially lateral root
development (Fig. 3-3 and Fig. 3-5). The chlorophyll levels in the starch mutants were
similar to the WT. However, among the starch deficient mutants, the pgm-1 mutant had
around 15% more chlorophyll than the adg1-1 mutant, although both mutants were
deficient in starch.
3.4.2 Plant Physiology
Starch content
The starch staining analysis on leaf showed that the WT had more starch present
in the leaves in afternoons compared to mornings. However, under either time point, the
WT did not contain as high a level of starch as the sex1-1 mutant did. The sex1-1 mutant
leaves contained more starch than all the other plant genotypes in both mornings and
afternoons (Fig. 3-6), but it showed decreased staining areas on the leaf in the afternoon,
which indicated that some starch was degraded into other metabolites. There was little to
no starch in the adg1-1 and the pgm-1 mutant leaves regardless of the time of the day.
The root tips were also stained for starch, which showed that both WT and sex1-1
mutants contained visible amounts of starch granules. However, in contrast to leaves
(Fig. 3-6), the sex1-1 mutant did not show significantly more starch in the root tip than
47
Chapter 3
the WT (Fig. 3-7). At the same time, there was a lack of starch in the root tips of the
adg1-1 and the pgm-1 mutants.
Carbon allocation
Results from the 11CO2 experiments showed that the starch mutants have altered
carbon relocation. The WT tended to transport more assimilated carbohydrates to the
belowground segments (roots + root exudates) in the afternoon than in the morning,
while both the adg1-1 and the pgm-1 mutants consistently showed 36% more of
assimilated carbon being transported into the belowground segments than the WT did in
both mornings and afternoons. The sex1-1 mutant transported 30% more assimilated
carbon to the belowground segments than the WT did in the morning but 17% less than
the WT in the afternoon.
The root exudation data indicated that 2% or lower of assimilated carbon was
secreted to the rhizosphere through plant roots and the amount of root exudation in all
types of Arabidopsis seedlings in this study exhibited a reduction in afternoons (Fig. 3-8).
Around 1% of the assimilated carbon in the WT was exuded to the rhizosphere. The sex11 mutant had a greater percentage, around 1.6% of assimilated carbon, being secreted
from the roots in the mornings, but only 0.8% in the afternoons. Among the starch
deficient mutants, the adg1-1 had a significantly higher amount, around 2%, of the
assimilated carbon exuded into the rhizosphere in the mornings (even higher than the
WT), and decreased to the WT level in the afternoons; whereas the pgm-1 mutant
exhibited the same levels of root exudates as the WT in both mornings and afternoons.
48
Chapter 3
3.4.3 Carbon Metabolism
The combination of 12C and 11C data showed that the starch mutants have
differing patterns of carbon partitioning from the WT.
Soluble sugars
Based on the 12C AM data (Fig. 3-9a), the sex1-1 mutant contained similar
amounts of all three soluble sugars as did the WT. The adg1-1 and the pgm-1 mutants
showed significantly more fructose and glucose present in the leaves than the WT, and
showed a comparable amount of sucrose to the WT. In the 12C PM data (Fig. 3-9b), the
adg1-1 and the pgm-1 mutants also showed higher amounts of fructose and glucose than
the WT as well as an increased amount of sucrose in the leaves compared to the WT.
Based on the 11C AM data (Fig. 3-9c), all types of Arabidopsis seedlings showed
a large fraction of fixed 11C assimilated into sucrose synthesis and relatively less into
fructose and glucose synthesis. The sex1-1 mutant had a trend of more 11C labeled
soluble sugars partitioned in the leaves than did the WT, but it was not statistically
significant. The adg1-1 and pgm-1 assimilated significantly more 11C into all three
soluble sugars, except that pgm-1 did not show as much 11C-sucrose as the adg1-1 mutant
did (16% vs. 21%). The 11C PM data showed a similar trend as the AM data with some
differences. Compared to the 11C AM data, all types of Arabidopsis in PM (Fig. 3-9d)
tended to have less 11C partitioned into 11C-fructose and 11C-glucose. As to sucrose, the
sex1-1 mutant showed decreased (from 17% to 10%), while the pgm-1 mutant showed
increased (from 16% to 21%) 11C partitioned into 11C-sucrose in PM than in AM. The
49
Chapter 3
WT and the adg1-1 did not show any notable changes in the amount of 11C partitioned
into sucrose from AM to PM.
Insoluble carbohydrates
The three starch mutants also showed differing patterns in carbon partitioning to
insoluble carbohydrates (Fig. 3-10). Around half of the fixed 11C was partitioned into
insoluble carbohydrates in the WT and it did not change from AM to PM. Compared to
the WT, all three starch mutants had a lower fraction of fixed 11C partitioned into
insoluble carbohydrates in the early mornings. This trend was also observed in the
afternoons.
3.4.4 Root Gravitropic Response
The sex1-1 mutant showed a root gravitropic response similar to the WT (Fig. 34). However, both of the starch deficient mutants exhibited significantly slower root tip
turning rates than the WT within the first 6 hours of the experiment, but a similar turning
rate as the WT at later times. The root turning rates did not stay constant. All Arabidopsis
seedlings showed two major increases in root turning rates during the experiment
regardless of the genotype; one was within the first three hours and the other was
between 6 to 9 hours into the experiment.
Root elongation rates were also measured along with the root gravitropic
response. Both the starch excess and the starch deficient mutants exhibited slower root
elongation rates than the WT. The root elongation rates fluctuated as did the root turning
rates. However, they were not in sync, but staggered with the increases occurring in root
turning rates.
50
Chapter 3
3.5 Discussion
3.5.1 Carbon Metabolism and Allocation
Our results show that the starch excess mutant and the starch deficient mutants
have notably altered patterns of carbon partitioning and carbon allocation compared to
the wild type. According to the conceptual model mentioned in Agtuca’s study [23], the
11
C data demonstrates snapshots of the dynamics of carbon flux, and is combined with
12
C analysis, which demonstrates the carbon pool size (such as the sugar pool size), to
gives a full picture of the fluctuation of carbon metabolism in both the starch mutants and
the WT.
Wild Type Arabidopsis
The combination of 12C and 11C data for the WT leaf tissue showed continuous
carbon influx (as soluble sugars) to the soluble sugar pools (Fig. 3-9c and d) during the
day but no change in the sugar pool sizes (Fig. 3-9a and b), suggesting that the
assimilated soluble sugars (such as fructose, glucose and sucrose) were used in the
downstream carbon metabolic pathways or translocated to other sink tissues in order to
keep the size of the sugar pools constant. Our insoluble carbohydrate data (Fig.3-10), root
allocation and exudation data (Fig.3-8) are consistent with the hypothesis.
A diurnal effect on carbon partitioning in the WT was observed. Less 11Cfructose and 11C-glucose was found in the leaves of the WT in the afternoon (Fig. 3-9d),
which suggests that the carbon portioning was likely re-directed. Fructose, glucose and
sucrose are under constant inter-conversions as shown in Equation 1, and the reaction
might be shifted towards sucrose in the afternoon, which means fructose and glucose
51
Chapter 3
were removed from their sugar pools. However, no increase was observed in the
downstream sugar metabolism, such as sucrose and starch synthesis in the WT (Fig. 3-9
and 3-10). The carbon allocation data (Fig. 3-8a) showed an increase in translocation of
carbohydrates to belowground, which suggests that more fructose and glucose were used
to synthesize sucrose in the afternoon, which was then transported to the roots of the WT.
The root allocation and exudation data showed that most of the carbohydrates
translocated to belowground were retained in the roots rather than secreted into the
rhizosphere. There was also a diurnal effect observed for carbon allocation to
belowground. More carbohydrates were translocated to belowground in the afternoon,
with more retained in the roots and a similar level in the root exudates compared to the
morning data.
sex1-1 Mutant
Previous research has shown that the sex1 (TC26) mutant exhibits a normal rate
of starch synthesis similar to the WT during the day, but a slower rate of starch
degradation at night [9, 30]. Over time the sex1 mutant tended to accumulate much more
starch in the leaves (Fig. 3-7). Our 11C data showed that the leaves of the sex1-1 mutant
exhibited a similar synthetic rate of the insoluble carbohydrates (mainly starch) as the
WT (Fig. 3-10). This impairment of starch degradation could lead to an imbalance
between starch synthesis and degradation, which may subsequently affect carbon
partitioning in the mutant. Zeeman’s study [9] showed that the sex4 mutant has elevated
amounts of fructose and glucose during the day, whereas the amount of sucrose was
comparable to the WT. However, prior to our study, there was no detailed sugar analysis
52
Chapter 3
of the sex1-1 mutant. Our data shows that the sugar pools in the sex1-1 mutant leaves
were not disturbed and exhibited similar levels of soluble sugars as the WT (Fig. 3-9a and
b). The amount of fixed 11C in leaves, however, tended to be partitioned more into the
fructose and glucose pools compared to the WT. The increased carbon influx during the
daytime did not change either the size of the soluble sugar pools or the amount of
assimilated insoluble carbohydrates, which suggests that the soluble sugars were likely
translocated rapidly, primarily as sucrose, from the source leaves to the other sink tissues,
such as the roots in this case. Compared to the WT, a higher amount of carbohydrates in
the belowground of the sex1-1 mutant was observed in the early morning (Fig 3-8), but
was significantly reduced in the afternoon, to levels below that of the WT. The
mechanism of the reduced amount of belowground carbohydrates in the sex1-1 mutant is
still unknown. A reduction of root exudates in the sex1-1 mutant in the afternoon was
also observed (Fig. 3-8b).
adg1-1 and pgm-1 Mutants
The adg1-1 and pgm-1 mutants are deficient in starch synthesis and only produce
around 2% of the starch that the wild type does [15, 16, 31, 32]. In our 11C study, a low
percentage of assimilated insoluble carbohydrates in the starch deficient mutants was also
observed (Fig. 3-10). Previous research has shown that the carbon in the pgm mutant was
redirected into soluble sugar synthesis, such as fructose, glucose and sucrose synthesis
[16]. In our study, the same trend was seen in both the adg1-1 and the pgm-1 mutants
(Fig. 3-9). In addition, we found that neither starch deficient mutant had elevated sucrose
in the early morning compared to the WT, but both showed an increase to twice the
quantity of the WT in the afternoon (Fig. 3-9a and 3-9b). Casper has shown that the
53
Chapter 3
soluble sugars in the pgm mutant were consumed rapidly at night [16] since there is little
starch available that can be used to re-generate sugars. The sugars are then regenerated
rapidly through photosynthesis in the early morning, refilling the sugar pools. Thus, it is
likely that the starch deficient mutants were completely depleted in sucrose overnight and
just started to synthesize and refill the sucrose pool, elevating the amount of sucrose to
the level observed in the WT at the time of measurement (Fig. 3-9c). In addition, it is
possible that large amounts of sucrose were exported into the roots at the same time (Fig
3-8a), which may have contributed to the relatively low sucrose levels observed in both
mutants in the early morning.
3.5.2 Root Morphology and Gravitropic Response
An imbalance in starch regulation not only alters carbon partitioning and
allocation, but also subsequently affects root morphology and root gravitropic responses.
We found that the starch deficient mutants tended to have a greater number of lateral
roots and total root area than the wild type (Fig. 3-3 and 3-5). This phenotype might be
the result of the increased carbohydrate translocation to the belowground segments
observed in these starch deficient mutants (Fig. 3-8). The carbohydrates translocated to
the belowground segments could be the excess soluble sugars produced by photosynthesis, or other carbon compounds serving as signaling molecules, such as auxin, or
both of them. Auxin has been shown to regulate lateral root growth. Thus, it is possible
that the auxin level in these starch deficient mutants is altered [33] and is responsible for
the increased lateral root growth, which creates a large sink for carbohydrates. However,
the mechanism of this elevated carbohydrate translocation to the roots in these starch
54
Chapter 3
deficient mutants is still unknown. This is worth studying in the future with the aim of
developing methods to improve root growth and increase root biomass.
The root gravitropic response has been shown to be related to the starch granules
in plant root tips. A lack of starch in plants can result in a loss of such response [33-35].
For example, the results of our root gravitropic response experiments showed that the
starch deficient mutants had reduced root turning angles (Fig. 3-4). The phosphor images
of the starch deficient mutants (Fig. 3-5) also showed “zig-zag” root growth patterns.
However, an over-accumulation of starch in plants, such as in the sex1-1 mutant, did not
show any impaired root gravitropic response. Our hypothesis is that starch is an initial
physical stimulus to root columbella cells in responding to the gravity and is not the only
factor that causes the gravitropic response. Our root gravitropic experiments showed that
the starch deficient mutants can eventually turn toward the ground even though they did
not show any starch granules in the root tips (only an observation). Followed by the
starch stimulus, a chemical signal was then induced to one side of the root cap cells to
expand. For example, the Cholodny-Went theory indicates that auxin plays a key role in
the root gravitropic response [36-38]. Such a mechanism explains: 1) why there was only
a significant difference between the starch deficient mutants and the WT at the beginning
of the gravitropic response, but not in the later stages (Fig. 3-4); and 2) why it showed a
staggered pattern between root turning rate and root growth rate in our root gravitropic
response experiments (Fig. 3-4).
55
Chapter 3
Figure 3-1. Brief summary of carbon fixation and downstream carbon metabolic pathway.
This figure also shows where the pathway is impaired for the three starch mutants used in the
studies. 1. sex1-1(Starch Excess1-1) mutant cannot degrade starch efficiently at night and
subsequently accumulates a lot of starch in the leaves; 2. adg1-1 (ADP-Glucose
pyrophosphorylase) mutant cannot transform Glc-1P into ADP-Glucose; 3. pgm-1
(phosphoglucomutase) mutant cannot transform Glc-6P into Glc-1P.
56
Chapter 3
A
B
Figure 3-2. The apparati used for 11CO2 pulsing and incubation. A: the tip box style
incubation chamber for sugar extraction assays; B: the rack style incubation chamber for
carbon relocation assays.
57
Figure 3-3. Comparison of root/shoot growth and chlorophyll content among the three starch mutants and WT.
For chlorophyll data, n= 10 seedlings, mean ± SE. For root morphology data, n= 10, mean ± SE. The statistics
show the comparisons between the mutants and the WT under corresponding conditions. See Table A2-2 in
Appendix A for tabular data.
Chapter 3
58
Figure 3-4. Comparison of root elongation and gravitropic responses among three starch mutants and
wild type Arabidopsis seedlings. n= 20 seedlings, mean ± SE. See Table A2-3 and A2-4 in Appendix
A for tabular data.
Chapter 3
59
Chapter 3
60
Figure 3-5. Carbon-11 phosphor imaging of the three starch mutants and the wild type.
Seedlings were exposed to the phosphor screen 1 hour after the 11CO2 pulsing.
Figure 3-6. Iodine staining assays of starch in three starch mutants and wild type Arabidopsis. The
assay was done both in the early morning and late afternoon, labeled as AM and PM.
Chapter 3
Figure 3-7. Iodine staining assay of starch in the root tips of three starch mutants and wild type
Arabidopsis.
61
Chapter 3
a.
b.
Figure 3-8. 11C-carbohydrate relocation to belowground and root exudation. n=8-14
seedlings. The figures show mean ± SE. The statistics show the comparisons between the
mutants and the corresponding wild type. See Table A2-8 in Appendix A for tabular
data.
62
Chapter 3
a
b
c
d
Figure 3-9. 12C and 11C sugar partitioning in the starch mutants and the wild type. a and b, C-12
analysis in AM and PM; c and d, C-11 analysis in AM and PM. Eighteen seedlings of each type of
Arabidopsis were combined for sugar extraction and the experiment was repeated 4-5 times. The
figures show mean ± SE. The statistics show the comparisons between the mutants and the
corresponding wild type. See Table A2-5 and A2-6 in Appendix A for tabular data.
63
Chapter 3
Figure 3-10. Percentage of total fixed 11C assimilated into insoluble carbohydrates.
18 seedlings of each type of Arabidopsis were combined for sugar extraction and the
experiment was repeated 4-5 times. The figures show mean ± SE. The statistics show
the comparisons between the mutants and the corresponding wild type. See Table A27 in Appendix A for tabular data.
64
Chapter 3
References
1.
Zimmermann, M., Ziegler, H., List of sugars and sugar alcohols in sieve-tube exudates.
Encyclopedia of Plant Physiology, NS, Springer, 1975. 1: p. 480-503.
2.
Chia, D.W., et al., Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and
other plant species. Planta, 2000. 211(5): p. 743-51.
3.
Slewinski, T.L. and D.M. Braun, Current perspectives on the regulation of whole-plant
carbohydrate partitioning. Plant Science, 2010. 178(4): p. 341-349.
4.
Stettler, M., et al., Blocking the metabolism of starch breakdown products in Arabidopsis
leaves triggers chloroplast degradation. Mol Plant, 2009. 2(6): p. 1233-46.
5.
Geigenberger, P., Regulation of starch biosynthesis in response to a fluctuating
environment. Plant Physiol, 2011. 155(4): p. 1566-77.
6.
Zeeman, S.C., S.M. Smith, and A.M. Smith, The diurnal metabolism of leaf starch.
Biochem J, 2007. 401(1): p. 13-28.
7.
Grennan, A.K., Regulation of starch metabolism in Arabidopsis leaves. Plant Physiol,
2006. 142(4): p. 1343-5.
8.
Orzechowski, S., Starch metabolism in leaves. Acta Biochimica Polonica, 2008. 55(3): p.
435-445.
9.
Zeeman, S.C. and T.A. Rees, Changes in carbohydrate metabolism and assimilate export
in starch-excess mutants of Arabidopsis. Plant, Cell & Environment, 1999. 22(11): p.
1445-1453.
10.
Schulze, W., et al., Growth and reproduction of Arabidopsis thaliana in relation to
storage of starch and nitrate in the wild-type and in starch-deficient and nitrate-uptakedeficient mutants. Plant, Cell & Environment, 1994. 17(7): p. 795-809.
11.
Graf, A., et al., Circadian control of carbohydrate availability for growth in Arabidopsis
plants at night. Proc Natl Acad Sci U S A, 2010. 107(20): p. 9458-63.
12.
Gibon, Y., et al., Adjustment of growth, starch turnover, protein content and central
metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short
photoperiods. Plant Cell Environ, 2009. 32(7): p. 859-74.
65
Chapter 3
13.
Smith, A.M. and M. Stitt, Coordination of carbon supply and plant growth. Plant Cell
Environ, 2007. 30(9): p. 1126-49.
14.
Cross, J.M., et al., Variation of enzyme activities and metabolite levels in 24 Arabidopsis
accessions growing in carbon-limited conditions. Plant Physiol, 2006. 142(4): p. 157488.
15.
Bahaji, A., et al., Arabidopsis thaliana mutants lacking ADP-glucose pyrophosphorylase
accumulate starch and wild-type ADP-glucose content: further evidence for the
occurrence of important sources, other than ADP-glucose pyrophosphorylase, of ADPglucose linked to leaf starch biosynthesis. Plant Cell Physiol, 2011. 52(7): p. 1162-76.
16.
Caspar, T., S.C. Huber, and C. Somerville, Alterations in Growth, Photosynthesis, and
Respiration in a Starchless Mutant of Arabidopsis thaliana (L.) Deficient in Chloroplast
Phosphoglucomutase Activity. Plant Physiol, 1985. 79(1): p. 11-7.
17.
Periappuram, C., et al., The plastidic phosphoglucomutase from Arabidopsis. A reversible
enzyme reaction with an important role in metabolic control. Plant Physiol, 2000. 122(4):
p. 1193-9.
18.
Yu, T.S., et al., The Arabidopsis sex1 mutant is defective in the R1 protein, a general
regulator of starch degradation in plants, and not in the chloroplast hexose transporter.
Plant Cell, 2001. 13(8): p. 1907-18.
19.
Smith, A.M., S.C. Zeeman, and S.M. Smith, Starch degradation. Annu Rev Plant Biol,
2005. 56: p. 73-98.
20.
Zeeman, S.C., et al., A starch-accumulating mutant of Arabidopsis thaliana deficient in a
chloroplastic starch-hydrolysing enzyme. Plant J, 1998. 15(3): p. 357-65.
21.
Trethewey, R.N. and T. ap Rees, A mutant of Arabidopsis thaliana lacking the ability to
transport glucose across the chloroplast envelope. Biochem J, 1994. 301 ( Pt 2): p. 44954.
22.
Streb, S., et al., The debate on the pathway of starch synthesis: a closer look at lowstarch mutants lacking plastidial phosphoglucomutase supports the chloroplast-localized
pathway. Plant Physiol, 2009. 151(4): p. 1769-72.
23.
Agtuca, B., Rieger, E., Hilger, K., Song , L., Ferrieri, R.A., Carbon-11 used to Study the
Physiological and Metabolic Basis for Hormonal Cross-Talk between Auxin and Salicylic
Acid in Maize (Zea mays L.) Root Development. (in review), 2013.
66
Chapter 3
24.
Kazan, K. and J.M. Manners, Linking development to defense: auxin in plant-pathogen
interactions. Trends Plant Sci, 2009. 14(7): p. 373-82.
25.
Woodward, A.W., Bartel, B., Auxin: Regulation, Action, and Interaction. Annals of
Botany, 2005. 95(5): p. 707-735.
26.
McSteen, P., Auxin and Monocot Development. Cold Spring Harbor Perspect. Biol 2010.
2: p. 1-17.
27.
Ferrieri, R.A., Wolf, A.P. , The chemistry of positron emitting nucleogenic atoms with
regards to preparation of labeled compounds of practical utility. Radiochimica Acta
1983. 34: p. 69-83.
28.
Klaus, R., Fisher, W., Hauck, H.E., Application of a thermal in situ reaction for
fluorometric detection of carbhhydrates on NH2-layers. chromatographia, 1990. 29: p.
467-472.
29.
Babst B.A., K.A.a.J.T., Biochemical partitioning of recently fixed carbon for studies of
rapid plant responses: Radiometabolite analysis of C-11 labeled nonstructural
carbohydrates. Plant Methods, 2012.
30.
Caspar, T., et al., Mutants of Arabidopsis with altered regulation of starch degradation.
Plant Physiol, 1991. 95(4): p. 1181-8.
31.
Gibon, Y., et al., Adjustment of diurnal starch turnover to short days: depletion of sugar
during the night leads to a temporary inhibition of carbohydrate utilization,
accumulation of sugars and post-translational activation of ADP-glucose
pyrophosphorylase in the following light period. Plant J, 2004. 39(6): p. 847-62.
32.
Niittyla, T., et al., A previously unknown maltose transporter essential for starch
degradation in leaves. Science, 2004. 303(5654): p. 87-9.
33.
Band, L.R., et. al., Root gravitropism is regulated by a transient lateral auxin gradient
controlled by a tipping-point mechanism. PNAS, 2012. 109: p. 4668-4673.
34.
Caspar, T. and B.G. Pickard, Gravitropism in a starchless mutant of Arabidopsis:
implications for the starch-statolith theory of gravity sensing. Planta, 1989. 177: p. 18597.
35.
Kiss, J.Z., R. Hertel, and F.D. Sack, Amyloplasts are necessary for full gravitropic
sensitivity in roots of Arabidopsis thaliana. Planta, 1989. 177: p. 198-206.
67
Chapter 3
36.
Cholodny, N., Wuchshormone und Tropismen bei den Pflanzen. Biol Zentralbl, 1927. 47:
p. 604-626.
37.
Went, F., Wuchsstoff und Wachstum. Rec Trav BotNeerl, 1928. 25: p. 1-116.
38.
Wolverton, C., Paya, A. M., Toska, J., Root cap angle and gravitropic response rate are
uncoupled in the Arabidopsis pgm-1 mutant. Physiol Plant, 2011. 141(4): p. 373-82.
68
Chapter 4: Effect of Fe status on Carbon Metabolism in Arabidopsis
4.1 Isotope Summary
Isotopes
11
C
Half-life
Decay Mode
Emax
β+ (99.8%) ,
0.96 MeV (β+)
EC (0.2%)
0.511 MeV annihilation photons (199.6%)
20.3 min
0.273 MeV (45%) and 0.466 MeV (53%) (β-);
59
Fe
44.6 d
β (100%), γ
-
1.099 MeV (57%) and 1.292 MeV (43%) (γ)
4.2 Background
Iron (Fe) is an essential mineral nutrient for plant growth and development. As a
co-factor for many vital enzymes [1, 2], including the cytochromes of the electron
transport chain [3], it is required for a wide range of biological functions, such as
chlorophyll synthesis [4, 5], and the maintenance of chloroplast structure and function [6,
7]. However, the bio-availability of Fe in the environment varies dramatically, which can
affect the normal growth of plants, especially crop species [8, 9]. Iron comprises around
5% of the earth’s crust, but in aerobic alkaline environments, Fe is very limited due to the
chemical form of Fe(III), mainly as oxyhydroxide polymers with extremely low solubility
in such environments. The bio-availability of Fe under these conditions is not sufficient
to meet plant needs. Plants under Fe deficient conditions can exhibit symptoms like
chlorosis in leaves (lack of chlorophyll) and inhibition of root growth. At the other
extreme, Fe can be readily available in excess for plants to take up in waterlogged soils
due to the low oxygen and low pH conditions [10]. Excessive Fe content is potentially
69
Chapter 4
toxic to plants because of the formation of reactive oxygen radicals in cells (Fenton
reaction), which can damage important cellular constituents, such as cell membranes.
Therefore, the Fe status in plants is tightly controlled. This behavior in plants is called
iron homeostasis [11].
The main mechanism to maintain Fe homeostasis is through the regulation of Fe
uptake by plant roots. To avoid excessive uptake of Fe, wetland plants have evolved
mechanisms for oxidizing ferrous Fe in the rhizosphere [12]. To cope with the Fe
limitation in aerobic soils, plants have developed two approaches, referred as Strategy I
and Strategy II (Fig. 4-1). Strategy II plants (graminaceous plants), such as grasses, rely
on the secretion of phyto-siderophores (PS) and subsequently take up the Fe(III)-PS
complex via a trans-membrane transporter, such as the Yellow Stripe1 (YS1) transporter
found in maize [13]. Strategy I plants (dicots, non-graminaceous plants), such as
Arabidopsis, rely on the induction of a series of physiological responses to assist in the
mobilization of the Fe compounds in the environment, such as ATPase-mediated
acidification of the rhizosphere [14], up-regulated activity of a plasma membrane-bound
ferric reductase (FRO2) [15, 16], and increased expression of an Fe(II) transporter (IRT1)
[15, 17, 18]. Impairment of these pathways can disturb the Fe homeostasis in plants and
subsequently affect plant growth and development. One Arabidopsis mutant (irt1-1) that
lacks IRT1 expression showed chlorosis and growth inhibition even with sufficient Fe
supplies [18]. On the other hand, the Arabidopsis mutant opt3-2 was recently found to
over-accumulate Fe in aerial tissues due to the constitutively up-regulated FRO2 and
IRT1 proteins [15, 19], known as the “Fe starvation” response, regardless of the Fe
availability in the environment.
70
Chapter 4
An imbalance of Fe status in plants not only can affect photosynthesis in leaves
due its contribution to chlorophyll synthesis, protein synthesis and electron-transfer chain
reactions [3-5, 7, 20], but also can affect carbon partitioning and allocation in both leaves
and roots. Carbon metabolism is vital for providing an energy source for plants,
contributing to plant skeleton development, and is also involved in metal transportation
[21, 22], plant long-distance signaling and root exudation. Fe deficiency stress is shown
to be associated with enhanced production of organic acids, particularly citric acid, which
is a known chelator of Fe(III) [21]. In the roots of Fe deficient plants, citrate
concentrations can be 3.7- to 8.8-fold higher than the plants with sufficient Fe supplies
[22]. Increases in organic acid concentrations in roots of Fe deficient plants are fairly
ubiquitous, and occur both in Strategy I and Strategy II plant species. Enhanced H+
exudation in Strategy I plant roots can occur in parallel with increased organic acid under
Fe deficient conditions [23]. Sugar metabolism is also found to be altered under Fe
deficient conditions in grapevine. Glucose-6-phosphate, fructose-6-phosphate, and 3phosphoglycerate content were found decreased as active Fe decreased in the plant cells
[24].
In this study, we investigated the relationship of Fe status and carbon metabolism
in two Arabidopsis mutants, opt3-2 and irt1-1. Both of these mutants have
malfunctioning Fe regulation systems, but in opposite ways as mentioned above. How the
disturbed Fe regulation affects carbon partitioning and allocation was studied using
radioactive isotopes of iron and carbon (59Fe and
11
C) to explore the dynamics of Fe
uptake, carbon metabolism and the cross-talk between them in the mutants and the wild
type (WT) under different Fe conditions.
71
Chapter 4
4.3 Materials and Methods
4.3.1 Materials
Bleach used for seed sterilization was purchased from Pure Bright (Columbia,
MO). The pH indicator, bromocresol purple, was purchased from Thermo Fisher
Scientific (New Jersey, USA). 59FeCl3 was purchased from Perkin Elmer (Waltham, MA,
USA). All other chemicals used in this study were purchased from Sigma Aldrich (St.
Louis, MO, USA).
4.3.2 Plant Growth Conditions
Seeds were surface-sterilized with a 35% bleach solution containing 0.1% Triton
X-100 for 15 min, and rinsed several times with sterile deionized water. Prior to
germination, seeds were placed at 4oC for 2–3 days to align the seed germination
times. Seeds were then germinated and grown in agar medium containing fullstrength modified [-Fe]-Hoagland’s solution (5 mM KNO3, 5 mM Ca(NO3)2, 2 mM
MgSO4, 1 mM NH4NO3, 0.5 mM KH2PO4, 46 nM H3BO3, 9 nM MnSO4, 0.77 nM
ZnSO4, 0.2 nM CuSO4 and 0.5 nM NaMoO4), 0.05% morpholinoethanesulfonic acid
(MES) and 0.5% agar. FeCl3 solution (50 µM) was added to the medium to ensure
normal seed germination. The pH of the medium was adjusted to 6.0 with 5 M NaOH
solution. Seedlings were grown at 22°C under 100 µmole m-2 s-1 fluorescent white
light with a 12 h (day)/12 h (night) photoperiod. One-week-old seedlings were transferred
to a new agar medium for various experimental purposes. For Fe treatment, FeCl3
solution was added to the nutrient medium to reach final concentrations of 9 µM (the Fe
72
Chapter 4
concentration present in a normal full-strength Hoagland’s solution) and 200 µM. For
root analysis, petri dish plates were placed vertically.
4.3.3 Chlorophyll Content Determination
Plants
were grown
under the
same conditions as
described
above in
the plant growth conditions. Various concentrations of FeCl3 (0, 9 and 200 µM) were
added into the growth medium. All the petri dish plates were placed horizontally and the
seedlings were grown for 2 weeks in the growth chamber. Eight seedlings were
selected from wild type (WT), opt3-2 and irt1-1 mutant plants grown under different Fe
treatments (0, 9 and 200 µM Fe3+). Individual seedlings were put into 1.5 ml Eppendorf
tubes and the chlorophyll was then extracted with 1 ml of 100% methanol. The extract
solutions (200 µl) were pipetted into 96-well plates and measured using a Bio-TEK plate
reader at 652, 665 and 750 nm. Chlorophyll content was calculated using the
following equations [25] :
Chl a = [16.29 x (A665 – A750)] – [8.54 x (A652 – A750)]
Chl b = [30.66 x (A652 – A750)] – [13.58 x (A665 – A750)]
Chl a+b = [22.12 x (A652 – A750)] + [2.71 x (A665 – A750)]
A652, A655 and A750 indicate the absorbance at 652, 655 and 750 nm respectively.
Chl a: Chlorophyll a; Chl b: Chlorophyll b; Chl a+b: Chlorophyll a and Chlorophyll b.
73
Chapter 4
4.3.4 Proton Concentration Measurements in the Rhizosphere
The pattern of pH change in the growth medium was determined by pH indicator
visualization [26]. Each agar plate contained 0.2 mM CaSO4, the pH indicator
bromocresol purple (0.006%) and 0.7% agar. The pH of the medium was adjusted to 6.0
with NaOH solution. Seeds were germinated in the agar media with modified [-Fe]Hoagland’s solution plus 50 µM FeCl3. The addition of Fe was to ensure normal seed
germination. One-week-old seedlings were transferred to agar media with various Fe
concentrations (0, 9 and 200 µM) and grown for one week before being transferred to the
agar medium containing the pH indicator. Photographs were taken 24 hours after the
transfer.
4.3.5 59Fe Uptake Assay
Preparation and Incubation
Seedlings were grown under the same conditions as mentioned in the Proton
Concentration Measurements. Individual seedlings were weighed before being
transferred into 1.5 ml centrifuge tubes filled with modified [-Fe]-Hoagland’s solution
plus the corresponding concentration of Fe. Centrifuge tubes were covered with parafilm,
leaving only the shoots of the plants outside (Fig. 4-2), to greatly reduce the possibility of
plant shoots contacting the radioactive incubation solution. Syringes and needles were
used to introduce the radioactive 59FeCl3 solution into the centrifuge tubes. Seedlings
were incubated in the radioactive 59FeCl3 solution for 2 hours before being harvested.
74
Chapter 4
Harvesting
Shoots were removed and placed in counting vials. Roots were removed from the
incubation solution, dried, and then placed into 1.5 mL centrifuge tubes filled with
cleaning solution and rinsed by gently vortexing the tubes. This action was repeated three
times, twice with the original incubation solution (without 59Fe) and once with 0.5 mM
CaCl2 solution (containing 0.05% MES, pH 6.0). In between each rinse, the roots were
dried with KimWipes. Finally, the roots were placed into counting vials. All the samples
were counted using liquid scintillation counting (LSC, Tri-Carb 2900TR, Perkin Elmer,
Waltham, MA, USA).
Figure 4-2. Experimental set-up for 59Fe uptake.
4.3.6 11CO2 production, pulsing and incubation apparatus
11
CO2 was produced via the 14N(p, α)11C nuclear transformation [27] from a 20 ml
target filled with high-purity nitrogen gas (400 mL @ STP) using 18 MeV protons from
the TR-19 (Ebco Industries Ltd, Richmond, BC, Canada) cyclotron at Brookhaven
National Laboratory (BNL), and captured on molecular sieves (4Å). The captured 11CO2
75
Chapter 4
was desorbed and quickly released into an air stream at 200 mL/min as a discrete pulse
for labeling the seedlings in different customized sealed incubation chambers (Fig 3-2,
Chapter 3). Seedlings within the chamber were pulse-fed 11CO2 for 1 minute, then chased
with normal air for the duration of the exposure.
Two types of customized apparatus were used as sealed systems during
11
CO2
pulsing and incubation of the seedlings. For sugar analysis, a square petri dish (without
its cover) with seedlings grown in the agar medium was placed within a sealed tip box
(Fig. 3-1A). For carbon relocation assays, 1.5 ml Eppendorf™ tubes with seedlings
grown in agar media were placed within a sealed custom fitted tube rack (Fig. 3-1B).
Both apparati had two connections, one for
11
CO2 introduction and one to serve as an
exhaust for the closed system. Fixed LED lights were placed on top of the tip box/ tube
rack to ensure normal photosynthesis. The 11CO2 experiments were conducted both in the
early morning (1 hour into the start of the photoperiod) and late afternoon (7 hours after
the AM experiment) as described in the Starch Staining Assay.
4.3.7 Tissue extraction and soluble sugar analysis
Seedlings for sugar analysis (wild type, opt3-2 and irt1-1 mutants) were grown on
square plates filled with modified [-Fe]-Hoagland’s solution plus various concentrations
of Fe treatments for 2-3 weeks. After
11
CO2 pulsing and 20 min of incubation, shoots
were harvested and immediately frozen in liquid nitrogen before being ground by hand in
pre-weighed and pre-chilled Eppendorf™ tubes. Four-times the mass of the powder (in
mg) was introduced as the volume of methanol (in µL) to the centrifuge tubes, vortexed
(VWR analog vortex mixer; Sigma-Aldrich Corp. St. Louis, MO, USA), and then
76
Chapter 4
sonicated (Branson Bransonic 32; Sigma-Aldrich Corp. St. Louis, MO, USA) at 0 C for
10 min. Additional vortexing was performed during this period to ensure complete
mixing. The tubes were centrifuged (Eppendorf Centrifuge 5424) for 2 min at 15,000 rpm
to separate the insoluble portion from the soluble one. The insoluble portion contained
mostly cell-wall polymers and starch. The soluble portion contained small soluble
compounds, including soluble sugars. These sugars were separated and analyzed by thin
layer chromatography (TLC) [28, 29]. Glass backed NH2-silica HPTLC-plates (200 µm,
w/UV254) were used for the sugar separation (Sorbent Technologies, Atlanta, GA,
USA). Plates were pre-spotted with 2 µL of sugar standards (including 4, 3, 2, 1, 0.5 and
0.2 mM glucose, sucrose and fructose) using a semi-automatic Linomat 5 sample
applicator (Camag Scientific Inc., Wilmington, NC, USA) for high precision of the spot
size and sample volume. Aliquots (2 µl) of leaf extract were also spotted. TLC plates
were developed using a mobile phase consisting of 75:25 acetonitrile/water (v/v). The
developed TLC plates were imaged using a phosphor imaging screen (FujiFilm BAS-MS
2040, GE Healthcare Biosciences, PA) to determine the yield of each radiolabeled sugar.
The plates were then heat-treated (200 C, 10 min) to initiate a chemical reaction of
individual sugars with the amino functionalized Si-support giving a fluorescence marker
detectable under long wavelength (365 nm) UV light [28]. Digital photographs were
taken of the fluorescent markers to provide a measure of the
12
C-sugar yields. Image
Quant TL software was used to analyze both the radiographic and the digital images to
determine the amount of carbon-11 (11C) and carbon-12 (12C) sugars.
Linear Log
regression was used for plotting standard curves generated from the 12C-sugar standards.
77
Chapter 4
4.3.8 Carbon Allocation
Seedlings for carbon allocation analysis were grown in square plates for one
week, as described in the Tissue Extraction and Soluble Sugar Analysis section.
Seedlings were transferred from growth plates into 1.5 ml Eppendorf™ tubes filled with
agar medium containing various concentrations of FeCl3 and allowed to grow for 2-3
weeks. After
11
CO2 pulsing and 1 hour incubation, the entire seedlings were removed
from the agar medium and cut into roots and shoots, then stored in respective vials filled
with methanol. Tissues were later counted in a NaI well-counter. The remaining tubes
containing agar medium were also placed individually into empty vials for counting,
which indicated the amount of 11C-labeled root exudates. This experiment was conducted
both in the early morning and late afternoon as mentioned in the
11
CO2 production,
pulsing and incubation apparatus.
The percentages of
11
C-phytosynthates transported to belowground and exuded
from the roots were calculated as follows:
A roots + A exudates
11
% of C-phytosynthates below =
× 100%
A shoots + A roots + A exudates
A exudates
11
% of C-phytosynthates exudates =
× 100%
A shoots + A roots + A exudates
A shoots, A roots and A exudates indicate the activities measured in the shoots, root tissues and
in the gel (root exudates).
78
Chapter 4
4.3.9 Phosphor imaging
After the Carbon Allocation assay, some of the seedlings were removed from the
1.5 ml Eppendorf™ tubes and exposed to a phosphor imaging screen (FujiFilm, BAS-MS
grade) for 20 seconds, which was then analyzed using a phosphor-imager (TyphoonTM
FLA 9000, GE Healthcare).
4.3.10 Statistical Analysis
Data was subjected to the Student t-test for unpaired samples assuming an
unequal variance. Statistical significance levels were assigned to the following rating
scale (*, p<0.05; **, p<0.01; ***, p<0.001).
4.4 Results
4.4.1 Plant Growth
The opt3-2 and irt1-1 mutants and the WT Arabidopsis seedlings were grown
under three different Fe conditions: no Fe, sufficient Fe (9 µM) and excess Fe (200 µM).
The shoot/root growth and the chlorophyll content of the mutants and the WT
Arabidopsis seedlings were compared.
The WT seedlings had low chlorophyll content and inhibited root growth under
Fe deficient conditions and showed increased chlorophyll content and improved
shoot/root growth under Fe sufficient and Fe excess conditions. There was no significant
change in the growth of the WT seedlings between Fe sufficient and Fe excess
conditions.
79
Chapter 4
The opt3-2 and irt1-1 mutants showed severe chlorosis and inhibited shoot/root
growth under Fe deficient condition, with the irt1-1 mutant seedlings more affected (Fig.
4-3). When sufficient Fe was present in the media, the opt3-2 mutant seedlings exhibited
increased levels of chlorophyll content and improved shoot/root growth compared to the
ones under Fe deficient conditions. The irt1-1 mutant showed equal levels of severe
chlorosis and inhibited shoot/root growth under the sufficient Fe conditions, as under the
Fe deficient conditions. However, under Fe excess conditions, the irt1-1 mutant
seedlings showed significant improvements in growth, with bigger shoots and longer
roots, in comparison to both the WT and the opt3-2 mutant. The chlorophyll content in
the irt1-1 mutant was also increased more under Fe excess conditions than under Fe
sufficient conditions, although it was still lower than observed with the WT and opt3-2
mutant.
4.4.2 Acidification of Rhizosphere through Root exudation
Each type of Arabidopsis seedling had a unique response to the varying levels of
Fe availability, with regard to the pH measurement assays of the root exudates. Seedlings
were originally grown in the media at pH 6.0. All three types of Arabidopsis seedlings
showed rhizosphere acidification (pH 5.0-5.5) around the roots under Fe deficient
conditions (Fig. 4-4B). With sufficient Fe3+ (9 µM) present in the media, all three types
showed increased pH (pH 6.5-7.0) in areas surrounding the roots, while the opt3-2 and
irt1-1 mutants continued to exhibit acidification around the roots (Fig. 4-4C). With
excess Fe3+ (200 µM) present in the medium, the WT and the opt3-2 mutant showed only
a slight change in the pH of root exudates, whereas the irt1-1 mutant exhibited low pH
80
Chapter 4
areas around the roots (Fig. 4-4D). The increasing pH observed around the roots is likely
due to NO3- supplied in the medium as a nitrogen source [30].
4.4.3 59Fe Uptake under Various Levels of External Fe Availability
The 59Fe uptake assay reports the amount of Fe taken up into a seedling within a
2-hour period (Fig. 4-5). With sufficient Fe (9 µM) available in the environment, the WT
tended to take up 45% more Fe than the opt3-2 and 52% more than the irt1-1 mutants.
With excess Fe present in the environment, both the WT and the opt3-2 mutants showed
around twice as much of the Fe uptake as the irt1-1 mutant did. The irt1-1 mutant
consistently showed low Fe uptake levels due to a lack of the IRT1 Fe transporter
regardless of the external Fe availability. Data for 59Fe uptake in the three Arabidopsis
genotypes under Fe deficient conditions are not shown here because the seedlings were
too weak to survive the 2 hours incubation.
4.4.4 Carbon Fixation, Metabolism and Allocation
The 11CO2 fixation data showed that there was no significant change in carbon
fixation levels in the WT seedlings when they were grown under Fe deficient or Fe
excess conditions (Fig. 4-6). The opt3-2 mutant had similar levels of carbon fixation as
the WT. However, the irt1-1 mutant showed a dramatic decrease in carbon fixation
compared to the WT when grown under Fe deficient conditions. For example, the irt1-1
mutant only fixed 19000 nCi of C-11 whereas the WT fixed around 45000 nCi of C-11.
As the amount of Fe present in the environment increased, the irt1-1 mutant showed
improved carbon fixation and reached a level similar to the WT.
81
Chapter 4
The levels of 11CO2 fixation in both mornings and afternoons were analyzed.
Under Fe deficient conditions, the carbon fixation levels of the three types of seedlings
did not change between the morning and afternoon. Under Fe sufficient and Fe excess
conditions, both the WT and the irt1-1 mutant showed decreased levels of carbon fixation
in the afternoon. Taking the carbon fixation under Fe sufficient conditions as an example,
the level in the WT decreased from 40000 nCi (AM) to 30000 nCi (PM), whereas the
level in the irt1-1 mutant decreased from 40000 nCi (AM) to 26000 nCi (PM). The opt32 mutant showed no change in carbon fixation level between morning and afternoon.
Our 11C metabolic data (Fig. 4-7) demonstrated that the opt3-2 and irt1-1 mutants
tended to assimilate 11C to high percentages of glucose in the leaves, especially under Fe
sufficient and Fe excess conditions, which was observed in both mornings and
afternoons. The opt3-2 mutant also showed a high percentage of glucose under Fe
deficient conditions during the morning (opt3-2 vs. WT, 6.9% vs. 5.4%) but not the
afternoon (opt3-2 vs. WT, 4.7% vs. 4.5%).
Sucrose was shown to be the primary form of soluble sugars and sucrose
synthesis showed a different pattern from glucose (Fig. 4-7B). The opt3-2 mutant
exhibited a similar percentage of sucrose as the WT did in the leaves regardless of the Fe
availability in the environment. However, the irt1-1 mutant showed a significantly lower
percentage of sucrose than the WT (irt1-1 vs. WT, 30.7% vs. 41.7% in the morning and
18.0% vs. 32.5% in the afternoon) under Fe deficient conditions and exhibited an
increase when more Fe was present in the environment, from 30.7% (Fe deficient) to
37.8% (Fe excess) in the morning and from 18.0% (Fe deficient) to 36.7% (Fe sufficient)
and 26.1% (Fe excess) in the afternoon. Under Fe excess conditions, the percentages of
82
Chapter 4
sucrose in the form of soluble sugars in the irt1-1 mutant reached the same levels as in
the WT (irt1-1 vs. WT, 37.8% vs. 33.7% in the morning and 26.1% vs. 24.0% in the
afternoon). Our results also showed that under Fe sufficient conditions, all three types of
Arabidopsis seedlings showed a higher percentage of sucrose in the afternoon (an
increase from 24.5% to 36.6% in the irt1-1 mutant), while under Fe deficient or Fe excess
conditions, a significantly reduced sucrose fraction was found in all the seedlings (a
decrease from 30.7% to 18.0% in the irt1-1 mutant under Fe deficient conditions).
Our carbon allocation data indicated that the WT had 2-4% of 11C-phytosynthates
transported belowground under Fe deficient conditions and exhibited an increase to
around 6% in the morning when more Fe was present in the environment (Fig. 4-8A).
Compared to the WT, the opt3-2 and the irt1-1 mutants exhibited high percentages of
11
C-phytosynthates in the belowground portion regardless of the Fe availability in the
environment. Our results also showed that all seedlings tended to transport relatively less
11
C-phytosynthates to the belowground in the afternoon (Fig 4-8B).
In addition, our root exudation data showed that the WT Arabidopsis had reduced
root exudation under Fe stress conditions, while the irt1-1 mutant constantly had
relatively high root exudation. The opt3-2 mutant behaved similarly to the WT, but only
showed increased root exudation under Fe excess conditions (Fig. 4-9A). Root
exudations were dramatically reduced in the afternoon for all the seedlings (Fig. 4-9B).
4.5 Discussion
Previous research has shown the Fe uptake ability and Fe regulation in the opt3-2
and irt1-1 mutants and all of the studies focus on Fe status in mature plants, with most of
83
Chapter 4
the results based on a long-term Fe accumulation process [15, 31]. Vert’s study [32] used
55
Fe as a radiotracer to track the Fe status in the irt1-1 mutant. However, the plants in his
study required 2 days of incubation, which is still a cumulative result. In our study, 59Fe
was used to investigate the total Fe uptake of the entire plant within hours. Also,
seedlings were used to test if they exhibit the same behavior as the mature plants.
Our 59Fe uptake results showed that, within the short incubation time window,
different Fe uptake abilities among mutants and the WT were observed, especially under
Fe excess conditions (Fig. 4-5). The irt1-1 mutant took up less 59Fe than the WT under
both Fe sufficient (6000 cpm/mg vs. 9000 cpm/mg) and Fe excess conditions (4000
cpm/mg vs. 10000 cpm/mg) (9 µM and 200 µM), whereas the opt3-2 mutant showed
similar levels of 59Fe as the WT did under all Fe treatments. Previous studies have shown
that the opt3-2 mutant plants over-accumulate Fe in the aerial tissues; however, this
phenotype was not seen in our 59Fe uptake experiments, where plants were only
incubated in the radioactive 59FeCl3 solution for 2 hours. This result indicates that Fe
over-accumulation phenotype of the opt3-2 mutant is very likely a result of long-term
regulation, rather than a kinetically rapid Fe uptake. (This behavior is also seen in heavy
metal accumulation in the opt3-2 mutant. See Chapter 2).
Iron plays key roles in many biological processes. Plant growth and development
can be significantly affected under long-term Fe stress (deficiency or toxicity). The most
common symptom under Fe deficiency is leaf chlorosis. This is because the precursor of
chlorophyll and heme synthesis is aminolevulinic acid (ALA), and the rate of ALA
synthesis is controlled by Fe [4]. Fe can also affect chloroplast development due its
contributions to many chloroplastic proteins and RNA syntheses, as well as its role in the
84
Chapter 4
electron transport chain. Under Fe deficient conditions, less Fe is present in the cells,
which can limit all of the bio-processes mentioned above and result in a chlorosis
phenotype.
The WT Arabidopsis seedlings in our study showed lower chlorophyll content
under Fe deficiency conditions than under Fe sufficient and Fe excess conditions (Fig. 43). However, our 11C data showed no significant change in carbon fixation in the WT
under any Fe treatments (Fig. 4-6). This result indicated a disconnection between
chlorophyll content and photosynthesis (i.e., it could be positively or negatively
correlated), which was also found in previous studies [33-35]. It is possible that the
decreased level of chlorophyll content in the WT was not significant enough to affect
carbon fixation.
The iron over-accumulation phenotype in the opt3-2 mutant can promote the
production of reactive oxygen species, which are radicals that can damage the regular
biological activities in plant cells, especially photosynthetic machinery in the
chloroplasts. Our 11C data showed that, compared to the WT, although the opt3-2 mutant
exhibited a relatively high level of carbon fixation under Fe sufficient conditions, a
decrease was observed under Fe excess conditions (Fig. 4-6). However, the Fe overaccumulation in the opt3-2 mutant did not inhibit chlorophyll synthesis in the mutant
(Fig. 4-3), which indicated that high Fe levels in plants is more likely to cause damage in
carbon fixation, rather than chlorophyll synthesis.
The irt1-1 mutant plants, with excess Fe present in the environment, showed
significant increases in chlorophyll content and shoot size, compared to itself under 9 µM
85
Chapter 4
Fe3+ treatment, although the irt1-1 mutant plants do not have an efficient Fe transporter
(IRT1) in roots [32, 36]. This result is consistent with the previous studies [32]. It was
shown that IRT1 is not the only iron transporter in root cells. IRT2 is also found in
Arabidopsis roots and up-regulated as part of the “Fe starvation” response although it is
not used as a major Fe transporter [37]. This contributes to the phenotype mentioned
above. The shoot growth of the irt1-1 showed a dramatic improvement, even having a
larger size than the WT under the same Fe conditions (Fig. 4-3). The mechanism for this
phenotype is still unclear. The carbon fixation level in the irt1-1 mutant showed a
positive correlation with chlorophyll content (Fig. 4-6), which supported that an Fe
deficient status in plants can significantly affect chlorophyll synthesis and subsequently
carbon fixation levels.
In addition to the effects on shoot growth, the internal Fe status can also affect
root growth. Fe deficiency is associated with inhibition of root elongation, increases in
the diameter of apical root zones, and abundant root hair formation [13, 38, 39]. Our
results showed that, under Fe deficient conditions, the irt1-1 mutant exhibited the most
severe inhibition of root growth compared to the other two types (Fig. 4-3). With addition
of 9 µM Fe3+, the root growth of all types of Arabidopsis seedlings improved notably,
with the irt1-1 mutant still exhibiting much shorter roots than the other two types. The
irt1-1 mutant showed dramatic improvements in root growth with an excess of Fe supply.
Like the shoot growth, the mechanism of this phenotype for the irt1-1 mutant is still
unknown.
Plant Fe status can also cause a redirection of carbon partitioning and carbon
allocation. Iron deficiency has generally been shown to cause increases in the organic
86
Chapter 4
acid concentrations, such as citrate, in roots, stem exudates and leaves of different plant
species [22, 40]. This shift in carbon usage can indirectly affect the carbon metabolic
pathways, such as sugar synthesis, which also can be affected by photosynthesis. In our
11
C experiments, glucose tended to be elevated in Fe-deficient plants (Fig. 4-7A),
whereas the sucrose level in the irt1-1 mutant was much lower than in the other two types
of Arabidopsis and this trend only occurred under low Fe concentrations (Fig. 4-7B).
This might be due to reduced carbon fixation ability (Fig. 4-6) as well as high levels of
carbon allocation to roots (Fig. 4-8).
Our 11C data showed that Fe status could alter carbon allocation and carbon
metabolism in plants and subsequently affect root exudation. Under Fe deficient
conditions, the opt3-2 and the irt1-1 mutants transported higher percentages of 11Cphotosynthates to the belowground compared to the WT. However, when more Fe was
added to the medium, this trend disappeared (Fig. 4-8A) This increased transport of
photosynthates to belowground in the mutants under Fe deficiency stress is possibly due
to a high demand for carbon in the roots for either enhanced root growth to reach out for
more Fe, or to synthesize Fe-stress related molecules, such as organic acids or chelators.
The data are not normalized by mass, so the increasing trend for the WT and the opt3-2
mutant under Fe sufficient and Fe excess conditions may be partially due to the
increasing root/mass ratio. However, the opt3-2 and irt1-1 mutants under Fe deficient
conditions showed high photosynthates present in roots even though they did not have
high root/shoot ratios (Fig. 4-3). Furthermore, our root exudation data indicated that the
irt1-1 had much higher amounts of carbohydrates exuded from the roots under Fe
deficiency stress. Also, the root exudation pH data showed that the irt1-1 exhibited
87
Chapter 4
rhizosphere acidification under Fe deficient conditions (Fig. 4-4). Therefore, the
combination of these two results suggests that, besides H+ exudation, the irt1-1 mutant is
likely to have elevated amounts of organic acids synthesized, which are transported to
roots and secreted to the rhizosphere to increase Fe acquisition ability. To verify the
hypothesis, root exudates need to be analyzed in the future.
In addition, we also found that the amount of carbon allocated to belowground
and secreted out of the roots was notably reduced in the afternoon for all types of
Arabidopsis under all Fe treatments. Therefore, this behavior is very likely a general
regulation pattern found in plants.
In this project, using 59Fe- and 11C as radiotracers has provided us an opportunity
to not only individually study the dynamic properties of the metabolism and allocation of
Fe and C within a short-time window, but also explore the relationships between Fe
homeostasis and carbon metabolism and allocation among the three types of Arabidopsis.
However, further investigations (root exudate analysis) and experimental repetitions (the
59
Fe uptake assays) are still needed in the future to better understand the cross-talk
between iron homeostasis and carbon metabolism.
88
Chapter 4
Figure 4-1. Two strategies for iron uptake in plants [41]. Strategy I is used by monocot and nongraminaceous plants and Strategy II is used by graminaceous plants. FRO, Ferric-chelate
Reductase; IRT, Iron Regulated Transporter; YS, Yellow Stripe.
89
Chapter 4
Figure 4-3. Comparison of root/shoot growth and chlorophyll content among the mutants and
WT. For chlorophyll data, n= 8 seedlings, mean ± SE. The statistics show the comparisons
between the mutants and the WT under corresponding conditions. The tabular data is listed in
Table 3-1 in Appendix A.
90
Chapter 4
91
Figure 4-4. Measurement of root exudate pH of Arabidopsis seedlings (wt, opt3-2and irt1-1)
grown under various concentrations of Fe treatments using a pH indicator. A, blank; B, without Fe;
C, with 9 µM Fe3+; D, with 200 µM Fe3+.
Chapter 4
92
*
Figure 4-5. 59Fe uptake under various levels of external Fe availability. n= 8-9 seedlings, mean ±
SE. The statistics show the comparisons between the mutants and the WT under corresponding
conditions. Tabular data is presented in Table A3-2 in Appendix A.
Chapter 4
93
A
B
Figure 4-6. Total 11CO2 fixation. The experiments were conducted both in the morning (A) and
afternoon (B). n= 6-8 seedlings. The statistics show the comparisons between the mutants and the WT
under corresponding conditions. Tabular data is listed in Table A3-3 in Appendix A.
Chapter 4
94
*
Figure 4-7. 11C-sugar partitioning in the leaves of three types of Arabidopsis seedlings. Fructose,
glucose and sucrose were extracted from leaf tissue and analyzed. This assay was conducted in both
morning (blue) and afternoon (orange). Eighteen seedlings of each type of Arabidopsis were
combined for the sugar extractions and the experiments were repeated 3-4 times. The figures show
mean ± SE. The statistics show the comparisons between the mutants and the corresponding wild
type. The same data plotted as bar graphs is shown in Figure A 3-2, A3-3 and A3-4 in Appendix A.
Tabular data is given in Table A3-4 in Appendix A.
Chapter 4
95
A
B
Figure 4-8. Allocation of 11C-photosynthates to the belowground. The experiments were conducted
both in the morning (A) and afternoon (B). The figures show mean ± SE, n= 6-8 seedlings. The
statistics show the comparisons between the mutants and the WT under corresponding conditions.
Tabular data is presented in Table A3-5 in Appendix A.
Chapter 4
96
A
B
Figure 4-9. Root exudation. The experiments were conducted both in the morning (A) and afternoon
(B). n= 6-8 seedlings. The statistics show the comparisons between the mutants and the WT under
corresponding conditions. Tabular data is given in Table A3-5 in Appendix A.
Chapter 4
References
1.
Fridovich, I., Superoxide radical: an endogenous toxicant. Annu. Rev. Pharmacol.
Toxicol., 1983. 23: p. 239-257.
2.
Hildebrand, D., Lipoxygenases. Physiol. Plant, 1989. 76: p. 249-253.
3.
Powers, L., et al., Structural features and the reaction mechanism of cytochrome oxidase:
iron and copper X-ray absorption fine structure. Biophysical Journal, 1981. 34(3): p.
465-498.
4.
Pushnik, J.C., Miller, G.W., Iron regulation of chloroplast photosynthetic function:
mediation of PSI development. J. Plant Nutr., 1989. 12: p. 407-421.
5.
Spiller, S.C., Castelfranco, A. M., Castelfranco, P. A., Effects of Iron and Oxygen on
Chlorophyll Biosynthesis: I. In Vivo Observations on Iron and Oxygen-Deficient Plants.
Plant Physiology, 1982. 69(1): p. 107-111.
6.
Abadia, J., Leaf responses to Fe deficiency: a review. Journal of Plant Nutrition, 1992.
15: p. 1699-1713.
7.
Pushnik, J., Miller, GW and Manwaring, JH, The role of iron in higher plant chlorophyll
biosynthesis, maintenance and chloroplast biogenesis. Journal of Plant Nutrition, 1984.
7: p. 733-758.
8.
Berman-Frank, I., et. al., Iron availability, cellular iron quotas, and nitrogen fixation in
Trichodesmium. . Limnol. Oceanogr., 2001. 46: p. 1249.
9.
Yamauchi, M., Rice Bronzing in Nigeria Caused by Nutrient Imbalances and its Control
by Potassium Sulfate Application. Plant Soil, 1989. 117: p. 275-286.
10.
Janiesch, P., Ecophysiological adaptations of higher plants in natural communities to
waterlogging. Miscellaneous Ecological responses to environmental stresses, 1991: p. 5060.
11.
Jeong, J. and M.L. Guerinot, Homing in on iron homeostasis in plants. Trends Plant Sci,
2009. 14(5): p. 280-5.
12.
Schmidt, W., Mechanisms and regulation of reduction-based iron uptake in plants. New
Phytol, 1999. 141: p. 1-26.
97
Chapter 4
13.
Romheld, V.M., H, Mobilization of Iron in the Rhizosphere of Different Plant Species.
Advances in in Plant Nutrition, 1986: p. 95-124.
14.
Toulon, V., et al., Role of apoplast acidification by the H+ pump. Planta, 1992. 186(2): p.
212-218.
15.
Stacey, M.G., et al., The Arabidopsis AtOPT3 protein functions in metal homeostasis and
movement of iron to developing seeds. Plant Physiol, 2008. 146(2): p. 589-601.
16.
Robinson, N.J., et al., A ferric-chelate reductase for iron uptake from soils. Nature, 1999.
397(6721): p. 694-7.
17.
Fox, T.C., et al., Direct Measurement of 59Fe-Labeled Fe2+ Influx in Roots of Pea
Using a Chelator Buffer System to Control Free Fe2+ in Solution. Plant Physiol, 1996.
111(1): p. 93-100.
18.
Eide, D., et al., A novel iron-regulated metal transporter from plants identified by
functional expression in yeast. Proc Natl Acad Sci U S A, 1996. 93(11): p. 5624-8.
19.
Stacey, M.G., et al., AtOPT3, a member of the oligopeptide transporter family, is
essential for embryo development in Arabidopsis. Plant Cell, 2002. 14(11): p. 2799-811.
20.
Lin, C.H. and C.R. Stocking, Influence of leaf age, light, dark, and iron deficiency on
polyribosome levels in maize leaves. Plant and Cell Physiology, 1978. 19(3): p. 461-470.
21.
de Vos, C.R., H.J. Lubberding, and H.F. Bienfait, Rhizosphere Acidification as a
Response to Iron Deficiency in Bean Plants. Plant Physiology, 1986. 81(3): p. 842-846.
22.
Abadía, J., et al., Organic acids and Fe deficiency: a review. Plant and Soil, 2002.
241(1): p. 75-86.
23.
Bienfait, H.F., Is there a metabolic link between H+ excretion and ferric reduction by
roots of Fe‐deficient plants?—a viewpoint. Journal of Plant Nutrition, 1996. 19(8-9): p.
1211-1222.
24.
Smith, B.R. and L. Cheng, Iron Assimilation and Carbon Metabolism in ‘Concord’
Grapevines Grown at Different pHs. Journal of the American Society for Horticultural
Science, 2007. 132(4): p. 473-483.
98
Chapter 4
25.
Porra, R.J., Thompson W.A., Kreidemann, P.E., Determination of Accurate Extinction
Coefficients and Simultaneous Equations for Assaying Chlorophylls a and b Extracted
with Four Different Solvents: Verification of the Concentration of Chlorophyll Standards
by Atomic Absorption Spectroscopy. 1989, Biochimica et Biophysica Acta. p. 348-389.
26.
Yi, Y. and M.L. Guerinot, Genetic evidence that induction of root Fe(III) chelate
reductase activity is necessary for iron uptake under iron deficiency. Plant J, 1996. 10(5):
p. 835-44.
27.
Ferrieri, R.A., Wolf, A.P. , The chemistry of positron emitting nucleogenic atoms with
regards to preparation of labeled compounds of practical utility. Radiochimica Acta
1983. 34: p. 69-83.
28.
Klaus, R., Fisher, W., Hauck, H.E., Application of a thermal in situ reaction for
fluorometric detection of carbhhydrates on NH2-layers. chromatographia, 1990. 29: p.
467-472.
29.
Babst B.A., K.A.a.J.T., Biochemical partitioning of recently fixed carbon for studies of
rapid plant responses: Radiometabolite analysis of C-11 labeled nonstructural
carbohydrates. Plant Methods, 2012.
30.
Marschner, H. and V. Römheld, In vivo Measurement of Root-induced pH Changes at the
Soil-Root Interface: Effect of Plant Species and Nitrogen Source. Zeitschrift für
Pflanzenphysiologie, 1983. 111(3): p. 241-251.
31.
Vert, G., et al., IRT1, an Arabidopsis transporter essential for iron uptake from the soil
and for plant growth. Plant Cell, 2002. 14(6): p. 1223-1233.
32.
Vert, G., et al., IRT1, an Arabidopsis Transporter Essential for Iron Uptake from the Soil
and for Plant Growth. The Plant Cell Online, 2002. 14(6): p. 1223-1233.
33.
Fleischer, W.E., The Relation Between Chlorophyll Content and Rate of Photosynthesis. J
Gen Physiol, 1935. 18(4): p. 573-97.
34.
Kirchhoff, W.R., et. al., Inheritance of a mutation influencing chlorophyll content and
composition in cowpea. Crop Science, 1989. 29: p. 105-108.
35.
Pettigrew, W.T., et al., Characterization of Canopy Photosynthesis of ChlorophyllDeficient Soybean Isolines. Crop Sci., 1989. 29(4): p. 1025-1029.
99
Chapter 4
36.
Vert, G.A., J.F. Briat, and C. Curie, Dual regulation of the Arabidopsis high-affinity root
iron uptake system by local and long-distance signals. Plant Physiol, 2003. 132(2): p.
796-804.
37.
Vert, G., J.F. Briat, and C. Curie, Arabidopsis IRT2 gene encodes a root-periphery iron
transporter. Plant J, 2001. 26(2): p. 181-9.
38.
Schmidt, W., Iron solutions: acquisition strategies and signaling pathways in plants.
Trends Plant Sci, 2003. 8(4): p. 188-93.
39.
Chaney, R.L., et al., Root hairs on chlorotic tomatoes are an effect of chlorosis rather
than part of the adaptive Fe‐stress‐response. Journal of Plant Nutrition, 1992. 15(10): p.
1857-1875.
40.
Fournier, J.M., E. Alcántara, and M.D. de la Guardia, Organic acid accumulation in roots
of two sunflower lines with a different response to iron deficiency. Journal of Plant
Nutrition, 1992. 15(10): p. 1747-1755.
41.
Ma, J.F., Plant Root Responses to Three Abundant Soil Minerals: Silicon, Aluminum and
Iron. Critical Reviews in Plant Sciences, 2005. 24(4): p. 267-281.
100
Chapter 5: Conclusions and Future Studies
Three plant biological studies using radiotracer methods have been discussed in
the previous chapters. This chapter aims to highlight the important findings in those
studies as well as to discuss the direction of future work.
In Chapter 2, heavy metal accumulation in the opt3-2 mutant Arabidopsis was
studied. We found that Pb2+ over-accumulates in opt3-2 mutant plants. Using radioactive
203
Pb to further explore the distribution and uptake dynamics of Pb ions in the opt3-2
mutant, we found that the over-accumulation of Pb is not due to a kinetically rapid Pb
uptake, but rather long-term accumulation. We also found that the up-regulated Fe
transporter IRT1 in the roots of the opt3-2 mutant does not transport Pb2+. It is likely that
other up-regulated transporters play a role in transporting Pb2+ in the opt3-2 mutant.
Further experimentation will need to be conducted to identify the up-regulated
transporters involved in Pb2+ trafficking. For example, the opt3-2/ transporter double
mutants (which have knocked-out the Atopt3 gene and those transporter genes) should be
grown under Pb treatment conditions. The plant growth of the double mutant plants
would be compared to the opt3-2 mutant to identify if any of the transporters are able to
transport Pb2+. Previous studies proposed that glutathione might play a key role in heavy
metal trafficking throughout the plant (unpublished data from David G. MendozaCozatl). The glutathione level in the opt3-2 mutant might be elevated. We conducted 35Sglutathione uptake assays (See Appendix A Fig. A1-8) in both the opt3-2 mutant and the
WT plants and found that the opt3-2 mutant tended to distribute the 35S-glutathione to all
leaves, while the WT plants clearly showed an asymmetric distribution of the
101
35
S-
Chapter 5
glutathione in the leave. However more replicates of this experiment will need to be
conducted in the future to verify the role of glutathione in heavy metal trafficking.
In Chapter 3, the carbon metabolism and translocation in Arabidopsis plants were
investigated using both 12C and 11C methods. The 11CO2 radiotracer, produced at BNL,
provided us the opportunity to explore the dynamics of carbon flux in plants, as well as
diurnal effects on carbon flux. According to the conceptual model mentioned in Agtuca’s
study [1], the 11C data demonstrates snapshots of the dynamics of carbon flux, and is
combined with 12C analysis, which demonstrates the carbon pool size (such as the sugar
pool size), to gives a full picture of the fluctuation of carbon metabolism in both the
starch mutants and the WT (Fig. 5-1).
11
C input flux
12
12
12
C
Glucose
Pool
C
Glucose
Pool
C
Glucose
Pool
col-0
sex1-1
(adg1-1 & pgm-1)
WT
Starch
Accumulate
Starch
Deficient
11
C
downstream
flux
Figure 5-1. A conceptual model used to interpretate 12C and 11C data.
102
Chapter 5
In this study, we found that starch regulation is essential not only for plant
growth, but also affects sugar metabolism (Fig. 5-1), carbon allocation, and root
exudation. Previous research has shown that plants interact with the rhizosphere through
root exudation, which affects the species of the micro-organisms living in the
rhizosphere; in turn, the altered rhizosphere will influence the plant growth [2, 3]. Our
11
C data showed that there were large amounts of carbon translocated to the belowground
of the plants (which might be in forms of sugars or auxin), and secreted into the
rhizosphere (especially with the starch deficient mutants). However, more experiments
are needed to identify the species of the carbon compounds translocated to belowground
and exuded into the rhizosphere. For example, an LC-MS method could potentially be
developed in the future to analyze the 11C-labeled carbon compounds in plant roots [4, 5].
In Chapter 4, the relationship between Fe status and carbon metabolism was
discussed. Two iron-transport mutants and the WT Arabidopsis were used for this study.
In this study, we found that the Fe status in plants affects the carbon fixation ability in
plant leaves, and also alters carbon partitioning (e.g., organic acid production), allocation,
and root exudation in plants. Diurnal effects on carbon metabolism and allocation were
observed in this study as well. Previous studies [6, 7] have shown that, under Fe deficient
conditions, plants tend to re-direct carbon to organic acid production in order to acquire
more Fe from the rhizosphere through chelation. In our
11
C data, the irt1-1 mutant
showed high levels of carbohydrates in the below-ground segments and root exudates
under Fe deficient conditions. Thus, it is possible that most of the carbon compounds
were in the form of organic acids. To validate this hypothesis, further experiments,
including as LC-MS analyses, are needed [4, 5].
103
Chapter 5
References
1.
Agtuca, B., Rieger, E., Hilger, K., Song , L., Ferrieri, R.A., Carbon-11 used to Study the
Physiological and Metabolic Basis for Hormonal Cross-Talk between Auxin and Salicylic
Acid in Maize (Zea mays L.) Root Development. (in review), 2013.
2.
Hirsch A.M., e.a., Molecular signals and receptors: controlling rhizosphere interactions
between plants and other organisms. Ecology, 2003. 84: p. 858-68.
3.
Bais, H.P., et. al., The Role of Root Exudates in Rhizosphere Interactions with Plants and
Other Organisms. Annu Rev Plant Biol, 2006. 57: p. 233-66.
4.
Chen, Z., et al., Confirmation and determination of carboxylic acids in root exudates
using LC-ESI-MS. J Sep Sci, 2007. 30(15): p. 2440-6.
5.
Oburger, E., et al., Evaluation of a novel tool for sampling root exudates from soil-grown
plants compared to conventional techniques. Environmental and Experimental Botany,
2013. 87(0): p. 235-247.
6.
Abadía, J., et al., Organic acids and Fe deficiency: a review. Plant and Soil, 2002.
241(1): p. 75-86.
7.
De Vos, C.R., H.J. Lubberding, and H.F. Bienfait, Rhizosphere Acidification as a
Response to Iron Deficiency in Bean Plants. Plant Physiology, 1986. 81(3): p. 842-846.
104
Appendix A: Supplementary Data
Appendix A
A1. Chapter 2
A1.1 Heavy Metal Treatments
Both the WT and the opt3-2 mutant plants were grown in media with different
metal treatments (Table A1-1). The purpose of these experiments was to investigate if the
opt3-2 mutant would show distinctive phenotypes under metal treatments compared to
the WT plants. Plants were grown under the same growth conditions as described in the
Plant Growth Conditions in Chapter 2.
Except under Pb, Hg and Cd treatments, we did not see dramatic differences
between the WT and the opt3-2 mutant plants when they were grown in the presence of
the other metals or the various treatments.
Table A1-1. Heavy metal treatments tested on the opt3-2 mutant plants.
Metal compounds
Concentration/µM
CdCl2
10
20
30
HgCl2
5
10
20
Pb(acetate)2
250
500
750
UO2(acetate)2
250
500
600/750
Na2HAsO4
30
60
120
FeCl3
50
100
200
GaCl3
50
100
200/250
105
Appendix A
A1.2 Seed Germination and Plant Root Growth under Different Heavy Metal
Treatments
For seed germination measurement, seeds were surface sterilized and cold-treated
as described in the Plant Growth Condition section in Chapter 2, before germination in
the MS agar media containing different concentrations of metals. The number of
germinated seeds was manually counted under a microscope during the first three days.
The germination percentage was calculated as shown in the equation below and in Figure
A1-1.
# of germinated seeds
Germination % =
x 100%
Total # of seeds
For root growth measurements, seeds were surface sterilized and cold-treated as
described in the Plant Growth Condition section in Chapter 2, before germination in halfstrength MS agar media. One-week-old seedlings were then transferred to agar media
containing different concentrations of the metals. After 1-1.5 weeks of a growing period,
pictures were taken using a camera and the root length was measured using the ImageJ
software. The percentage of root elongation inhibition (compared to the WT under no
metal treatment) was calculated as shown in the equation below and shown in Figure A12:
|Lmetal-Lcontrol|
Root elongation inhibition % =
x 100%
Lcontrol
Lmetal: root length of the WT or the opt3-2 mutant plants grown under metal treatments.
Lcontrol: root length of the WT or the opt3-2 mutant plants grown without metal treatments.
106
Appendix A
(Continued to the next page)
107
Appendix A
Figure A1-1. Comparisons of seed germination rates under different heavy
metal treatments within the first three days. The number after the each heavy
metal symbol represents the concentration in µM of that heavy metal. n=1.
Figure A1-2. Comparisons of root elongation under different heavy metal
treatments. The number after the each heavy metal symbol represents the
concentration in µM of that heavy metal. n=8-12 seedlings, mean ± SE. *, p<0.05;
**, p<0.01; ***, p<0.001.
108
Appendix A
A1.3 Characterization of RBS
We have tried to use fluorescence imaging technology to investigate the
distribution of Hg2+ in plant tissues. Previous studies have shown that Rhodamine B
Thiolactone (RBS, Scheme A1-1) is a promising Hg2+ fluorescence probe, which has
enhanced fluorescence at around 585 nm ( λex= 530 nm) when interacting with Hg2+.
RBS was shown to be selectivefor Hg2+ detection in the presence of other metal ions
commonly seen in plant tissues (Scheme A1-2) [1]. RBS was synthesized based on Wen
Shi’s method [2] and characterized using both 1H-NMR (Fig. A1-3) and 13C-NMR (Fig.
A1-4). Before testing on plant tissues, the RBS solution was evaluated with several
concentrations of HgCl2 solutions (Fig. A1-5).
Scheme A1-1. Structure of RBS.
109
Appendix A
Scheme A1-2. Possible reaction mechanisms of RBS with HgCl2.
110
Figure A1-3. 1H-NMR of RBS (300 MHz), dissolved in CDCl3.
Appendix A
111
Figure A1-4.
C-NMR. of RBS (500 MHz), dissolved in CDCl3.
13
Appendix A
112
Figure A1-5. Fluorescence Spectra of RBS with different concentrations of Hg2+. RBS1 and RBS2 are the same
molecule made in two separate trials. The excitation wave length is 560nm with slit openness of ex1.5 and
em1.5 except that RBS2_Hg0.5 also used ex3.0 and em3.0. The shift in the green curve is probably due to a
different interaction between RBS and Hg under low Hg concentration.
RBS1_Hg20
RBS2_Hg20
RBS2_Hg0.5_3.0
RBS2_Hg0.5
Appendix A
113
Appendix A
A1.4 Plant physiology (including opt3-2 mutant, irt1-1 mutant and wild type)
The irt1-1 mutant was added into this experiment to make a comparison with the
opt3-2 mutant and the wild type Arabidopsis. Plants were grown using the same method
described in Plant Growth Conditions in Chapter 2. The only difference is that an extra
50 µM of Fe3+ was added into the media to keep the normal growth of the irt1-1 mutant.
The results showed that the growth of the irt1-1 mutant was inhibited under high
concentrations of Pb/Hg treatment, which is at a similar level to that observed with the
opt3-2 mutant. This result indicates that IRT1-1 does not play a key role in Pb/Hg
trafficking.
Figure A1-6. Comparison of shoot growth of the opt3-2, irt1-1 mutants and the wild type
Arabidopsis with various concentrations of Pb/Hg treatments. 50 µM Fe3+ was added to all the
media to ensure a healthy growth of the irt1-1 mutant.
114
Appendix A
Figure A1-7. Comparison of the root growth for the opt3-2, irt1-1 mutants and the wild type
Arabidopsis under various concentrations of Pb/Hg treatments. 50 µM Fe3+ was added in all the
media to ensure a healthy growth of the irt1-1 mutant.
115
Appendix A
A1.5 Glutathione uptake and distribution in the opt3-2 mutant
In this experiment, one-month old WT and opt3-2 mutant plants were used
(grown in soil). A load leaf was first abraded with a fabric wheel to generate holes on the
leaf surface so that the incubation solution (Table A1-2) can be taken up through the
load leaf. Then the load leaf was placed into a manually cut 1.5 ml centrifuge tube (Fig.
A1-8). After 24 hours of incubation, the load leaf was removed from the plant. The entire
shoot was then removed from the roots and dissected into individual leaves (Fig. A1-9).
The leaves were then exposed to the phosphor screen for 10 hours to acquire the
phosphor images. For solution preparation and experimental parameters, please refer to
Table A1-2.
Figure A1-8. Illustration of load leaf incubation in the [35S]-glutathione
containing solution.
116
Appendix A
Figure A1-9. 35S-glutathione uptake through leaf administration and
distribution patterns in the opt3-2 mutant (right) and the WT (left). Red
arrows indicate the administration leaf, which are removed in the middle
and the bottom pictures.
117
12 h (day)/12 h (dark) (ON: 8 AM; OFF: 8 PM)
9:36am
24 hours
10 hours
25 mM GSH in DI water
[S-35] GSH stock
Tris buffer (pH 7.5)
10 µl stock GSH + 20 µl hot GSH stock + 470 µl Tris buffer = 500 µl total
30 µCi
Light cycle
Start time
Incubation
Exposure
Solutions used
Final incubation solution
Activity applied per plant
Table A1-2. Experimental parameters for 35S-glutathione uptake assay.
Appendix A
118
Appendix A
Table A1-3. Data of root / shoot growth of plants under Hg or
Pb treatments.
WT
Treatments
Control
avg.
SE
Hg10
avg.
SE
Hg20
avg.
SE
Pb500
avg.
SE
Pb750
avg.
SE
Shoots
(mg)
22.43
21.93
25.37
20.80
22.63
0.10
17.63
20.43
20.00
15.33
18.35
0.12
16.50
14.53
13.93
11.73
14.18
0.10
14.40
14.90
17.47
14.33
15.28
0.07
8.80
8.47
10.03
10.37
9.42
0.05
Roots
(mg)
8.37
8.67
9.13
9.07
8.81
0.02
6.47
8.33
7.93
5.23
6.99
0.07
5.40
5.07
4.20
4.80
4.87
0.03
5.53
7.10
7.97
4.77
6.34
0.07
1.97
1.90
2.13
2.53
2.13
0.01
opt3-2
Shoots
Roots
(mg)
(mg)
15.50
8.13
16.57
10.77
13.40
8.67
14.80
8.60
15.07
9.04
0.07
0.06
14.20
5.50
10.00
3.73
11.20
4.53
13.17
5.57
12.14
4.83
0.09
0.04
8.57
3.20
10.73
2.87
8.33
2.27
9.60
2.77
9.31
2.78
0.05
0.02
10.20
3.83
11.33
4.17
11.23
2.77
10.20
3.17
10.74
3.48
0.03
0.03
6.73
0.77
6.67
1.23
5.80
0.77
5.83
0.97
6.26
0.93
0.03
0.01
119
Appendix A
Table A1-4. Data of chlorophyll content
Treatments
Control
avg.
SE
Hg10
avg.
SE
Hg20
avg.
SE
Pb500
avg.
SE
Pb750
avg.
SE
Chl (µg/ g f.w.)
WT
opt3-2
607
626
609
571
690
545
755
776
569
665
36
581
809
927
846
577
748
72
412
635
556
639
470
542
45
670
698
567
544
606
617
30
431
430
524
544
405
467
28
617
42
667
508
659
342
482
531
61
463
400
411
400
305
374
392
21
587
512
506
719
581
49
299
380
224
405
327
41
120
10574.49
194.73
120.18
92.21
3.06
SE
2588.83
2709.01
21.92
346.09
15.79
18.86
7274.48
avg.
7312.67
19.09
10379.76 10769.23
1625.39
67.14
2829.19
1546.60
75.16
438.29
253.88
1775.52
1.46
330.28
SE
opt3-2
480.61
7293.57
11.55
1649.17
10.26
405.44
16.59
Rosette Leaves
Pb Treatment
Rosette Leaves
Influorescence Stem
13.33
14.91
Control
Influorescence Stem
avg.
Plant
Types
WT
Table A1-5. ICP-MS Pb data (ppb).
Appendix A
121
40.10
1.60
SE
38.50
avg.
41.70
1.75
SE
opt3-2
40.90
42.65
44.40
100.20
4.25
154.45
11.35
150.20
158.70
111.55
122.90
Control
Influorescence Stem
Rosette Leaves
avg.
Plant
Types
WT
Table A1-6. ICP-MS Hg data (ppm).
63.55
53.00
16.00
76.90
10.55
92.90
60.90
74.10
756.70
113.95
830.45
23.90
944.40
716.50
732.80
708.90
Hg Treatment
Influorescence Stem
Rosette Leaves
Appendix A
122
Appendix A
Table A1-7. 203Pb distribution in opt3-2 mutant and WT plants.
Date of
Expt.
Tissue
Type
Normalized Activity
(cpm/g f.w.)
Percentage of 203Pb
transported to
shoots/roots
4/25/2013
As
Ar
Bs
Br
As
Ar
Bs
Br
As
Ar
Bs
Br
As
Ar
Bs
Br
55765
20443988
7156
24856210
39861
19817720
49951
14045935
39578
19660563
22788
17682114
45944
39931622
20454
14561445
0.27
99.73
0.03
99.97
0.20
99.80
0.35
99.65
0.20
99.80
0.13
99.87
0.11
99.89
0.14
99.86
4/26/2013
4/27/2013
4/28/2013
Notes: As, WT shoots; Ar, WT roots; Bs, opt3-2 shoots; Br, opt3-2 roots.
Total 203Pb
Activity
(entire plant,
cpm/g f.w.)
20499753
24863366
19857581
14095886
19700141
17704902
39977566
14581899
123
Appendix A
A2. Chanpter 3
Table A2-1. List of sugars
Name
Molecular formula
M. W
Fructose
C6H12O6
180.2
Glucose
C6H12O6
180.2
Sucrose
C12H22O11
342.3
Maltose
C12H22O11
342.3
ADP-Glucose
C16H23N5O15P2
589.3
UDP-Glucose
C15H24N2O17P2
566.3
Fructose 6phosphate
C6H13O9P
260.1
structure
124
Appendix A
Figure A2-1. Carbon fixation in the three starch mutants and the wild type
Arabidopsis. n= 7-14, mean ± SE. *, p≤0.05; **, p≤0.01; ***, p≤0.001.
Table A2-2. Data of Chlorophyll content.
Replicates
avg.
SE
Chlorophyll Content (µg/g f.w.)
WT
sex1-1
adg1-1
pgm-1
504
488
321
408
485
326
413
600
466
512
398
479
539
469
574
522
568
482
334
531
480
466
347
550
532
574
432
599
450
334
383
504
481
419
516
611
341
418
523
593
485
449
424
540
20
24
27
20
125
Time
col-0
Point (hr) Avg. Length(mm)
0
8.5
3
8.9
6
11.1
9
11.4
12
12.2
21
13.9
24
14.2
SE
0.3
0.3
0.4
0.4
0.4
0.5
0.5
sex1-1
Avg. Length(mm)
5.1
5.5
6.6
7.0
7.3
7.9
8.3
SE
0.3
0.3
0.4
0.4
0.5
0.5
0.6
adg1-1
Avg. Length(mm)
4.3
4.6
5.8
6.0
6.0
7.0
6.9
SE
0.2
0.3
0.3
0.3
0.4
0.4
0.4
pgm-1
Avg. Length(mm)
3.8
3.9
5.1
5.3
6.0
6.1
6.2
Table A2-3. Comparison of root elongation among three starch mutants and wild type Arabidopsis seedlings.
SE
0.2
0.3
0.3
0.3
0.4
0.4
0.4
Appendix A
126
Time
col-0
Point (hr) Avg. Angle(degree)
0
1.4
3
37.0
6
48.0
9
64.5
12
73.1
21
83.7
24
84.5
SE
0.4
3.4
3.2
2.6
3.0
2.6
2.3
sex1-1
Avg. Angle(degree)
2.7
46.8
51.9
65.3
69.9
81.5
83.6
SE
1.0
1.7
2.9
2.4
1.4
1.4
1.7
adg1-1
Avg. Angle(degree)
2.8
18.3
19.5
31.9
39.5
60.8
64.7
SE
1.9
3.1
2.9
3.7
3.4
3.8
4.7
pgm-1
Avg. Angle(degree)
5.2
20.1
22.0
33.7
45.1
57.5
60.5
Table A2-4. Comparison of root gravitropic responses among three starch mutants and wild type Arabidopsis seedlings.
SE
2.0
3.7
4.1
4.5
4.0
3.4
4.5
Appendix A
127
Fructose
Glucose
Sucrose
Fructose
Glucose
Sucrose
Sugars
C
(%)
1.4
1.8
13.2
0.5
0.9
11.2
col-0
0.2
0.1
1.4
0.1
0.1
0.5
SE
sex1-1
C
(%)
3.6
4.0
17.0
1.3
1.5
10.0
11
0.8
1.1
2.0
0.2
0.2
1.3
SE
adg1-1
C
(%)
7.4
9.4
20.9
3.6
4.1
20.2
11
1.2
0.8
2.7
0.7
0.6
2.8
SE
pgm-1
C
(%)
8.5
11.2
15.5
4.9
5.7
20.9
11
1.8
1.4
1.2
0.6
0.7
1.9
SE
18 seedlings of each type of Arabidopsis were combined for sugar extractions and the experiments were repeated 4-5
times. The figures show mean ± SE.
PM
AM
Time
11
Table A2-5. Percentage of 11C sugar partitioning in the starch mutants and the wild type
Appendix A
128
PM
AM
Time
12
C
(µmoles/g f.w.)
0.32
0.57
0.37
0.30
0.50
0.43
Sugars
Fructose
Glucose
Sucrose
Fructose
Glucose
Sucrose
col-0
0.05
0.05
0.04
0.06
0.04
0.06
SE
sex1-1
0.39
0.62
0.38
0.32
0.57
0.40
C
(µmoles/g f.w.)
12
Table A2-6. 12C sugar partitioning in the starch mutants and the wild type
0.06
0.10
0.04
0.06
0.06
0.04
SE
adg1-1
0.77
1.42
0.57
0.56
1.18
0.82
C
(µmoles/g f.w.)
12
0.12
0.24
0.07
0.09
0.09
0.10
SE
pgm-1
0.66
1.45
0.46
0.60
1.40
0.72
C
(µmoles/g f.w.)
12
0.10
0.18
0.06
0.14
0.34
0.13
SE
Appendix A
129
Appendix A
Table A2-7. Percentage of total fixed 11C assimilated into insoluble
carbohydrates.
AM
Plants
WT
sex1-1
adg1-1
pgm-1
Carbohydrates Fraction
of 11C
Insoluble
0.53
Extract
0.47
Insoluble
0.44
Extract
0.56
Insoluble
0.36
Extract
0.64
Insoluble
0.34
Extract
0.66
PM
SE
0.04
0.04
0.03
0.03
0.02
0.02
0.07
0.07
Fraction
of 11C
0.57
0.43
0.53
0.47
0.44
0.56
0.41
0.59
SE
0.02
0.02
0.05
0.05
0.05
0.05
0.01
0.01
Table A2-8. 11C-carbohydrate relocation to belowground and root exudates.
AM
PM
C transported
to
belowground (%)
12.4
10.2
14.9
16.5
11
Plants
11
C transported to
belowground (%)
9.9
13.2
15.4
15.6
WT
sex1-1
adg1-1
pgm-1
SE
1.0
1.0
1.1
1.3
AM
Plants
WT
sex1-1
adg1-1
pgm-1
11
C exuded from roots
(%)
1.1
1.6
2.1
1.0
SE
0.5
0.6
1.0
1.2
PM
SE
0.2
0.2
0.3
0.1
11
C exuded from roots
(%)
1.0
0.8
1.0
1.1
SE
0.1
0.1
0.1
0.1
130
avg.
SE
Replicates
Chlorophyll
(µg/g f.w.)
WT
451
380
395
471
406
570
484
629
473
31
Fe0
opt3-2
516
408
510
463
443
393
513
566
477
21
Table A3-1. Chlorophyll content.
irt1-1
146
210
256
250
691
214
373
192
292
62
WT
1707
685
834
642
948
762
865
846
911
119
Fe9
opt3-2
783
739
613
468
744
742
908
826
728
48
irt1-1
255
245
226
215
238
162
225
232
225
10
WT
911
997
690
882
923
829
740
1128
888
49
Fe200
opt3-2
900
841
1080
976
857
1033
885
1107
960
37
irt1-1
798
603
741
634
574
703
684
585
665
28
Appendix A
A3. Chapter 4
131
avg.
SE
Replicates
Fe-59 uptake
(cpm/mg f.w.)
Table A3-2.
3810
618
WT
3968
5706
4881
2473
4205
1627
4870
1298
Fe0
opt3-2
7655
3014
3337
9954
3389
1872
2645
415
irt1-1
3596
3655
2128
1931
1231
3328
3710
1660
WT
1474
2594
1981
2652
1607
11952
3102
603
AM
Fe9
opt3-2
2254
2612
2192
3009
5442
14445
3246
880
irt1-1
2556
1357
787
5510
3184
6080
7178
831
WT
8960
4735
5683
5628
8807
9253
5108
1391
Fe200
opt3-2
9171
8214
6633
711
3666
2250
5089
1348
irt1-1
3239
3638
5217
1358
6176
10906
Fe uptake under various levels of external Fe availability.
59
WT
4496
3762
8812
6404
6760
6751
3293
3583
1860
5080
738
Fe0
opt3-2
9445
4418
6818
6348
6936
5561
3125
5864
5663
6020
585
irt1-1
5619
2392
2403
5046
8180
8483
3439
3476
2848
4654
786
WT
10025
13194
9640
12459
12891
11533
4383
5962
1241
9037
1411
PM
Fe9
opt3-2
2765
5439
4764
4386
11845
14705
6094
2972
3138
6621
1529
irt1-1
3648
3109
3881
8191
5015
6612
7093
13403
1010
5773
1207
10517
2494
WT
8108
13182
15495
8397
1037
12545
22753
2620
Fe200
opt3-2
2308
10502
14838
3826
13719
10256
9090
14764
2232
9060
1711
irt1-1
3265
2985
3697
7074
1536
10800
2249
7816
447
4430
1130
Appendix A
132
Appendix A
Figure A3-1. Total 11CO2 fixation. The experiments were conducted both
in the morning (A) and afternoon (B). n= 6-8 seedlings. The statistics
show the comparisons between the mutants and the WT under
corresponding conditions. Standard errors were calculated, but not shown
here. No statistically significant differences were found in this set of data.
Error bars were omitted for clarity.
Table A3-3. Total 11CO2 fixation.
Fe
conditions
Fe0
Fe9
Fe200
Plants
WT
opt3-2
irt1-1
WT
opt3-2
irt1-1
WT
opt3-2
irt1-1
AM
Total 11C
fixation
(nCi)
44698
42813
18747
38956
44785
37843
44144
34924
45094
PM
SE
Total 11C
fixation (nCi)
SE
2936
2501
2097
2287
3445
3504
4185
3322
6024
38701
35074
18462
31099
45352
26015
35266
30660
28585
6616
3386
2275
2285
6403
1686
3620
2668
2069
133
Figure A3-2. 11C-sugar partitioning in the leaves of three types of Arabidopsis seedlings. Fructose was extracted from
leaf tissue and analyzed. This assay was conducted in both the morning and the afternoon . Eighteen seedlings of each
type of Arabidopsis were combined for sugar extractions and the experiments were repeated 3-4 times. The figures show
mean ± SE. The statistics show the comparisons between the mutants and the corresponding wild type.
Appendix A
134
Figure A3-3. 11C-sugar partitioning in the leaves of three types of Arabidopsis seedlings. Glucose was extracted from
leaf tissue and analyzed. This assay was conducted in both the morning and the afternoon . Eighteen seedlings of each
type of Arabidopsis were combined for sugar extractions and the experiments were repeated 3-4 times. The figures
show mean ± SE. The statistics show the comparisons between the mutants and the corresponding wild type.
Appendix A
135
Figure A3-4. 11C-sugar partitioning in the leaves of three types of Arabidopsis seedlings. Sucrose was extracted
from leaf tissue and analyzed. This assay was conducted in both the morning and the afternoon . Eighteen seedlings
of each type of Arabidopsis were combined for sugar extractions and the experiments were repeated 3-4 times. The
figures show mean ± SE. The statistics show the comparisons between the mutants and the corresponding wild type.
Appendix A
136
Appendix A
Table A3-4. 11C-sugar partitioning in the leaves of three types of Arabidopsis seedlings.
Time
Sugars
WT
Percentage of
Soluble Sugars
SE
11
Fructose
AM Glucose
Sucrose
Fructose
PM Glucose
Sucrose
Time
Sugars
( C)
4.8
5.4
41.7
3.9
4.5
32.5
WT
Percentage of
Soluble Sugars
Fructose
AM Glucose
Sucrose
Fructose
PM Glucose
Sucrose
Time
Sugars
WT
Percentage of
Soluble Sugars
0.3
0.3
2.6
0.8
0.9
2.3
SE
Fructose
AM Glucose
Sucrose
Fructose
PM Glucose
Sucrose
( C)
6.2
6.9
40.2
3.5
4.7
34.7
Fe9
opt3-2
Percentage of
Soluble Sugars
0.2
0.2
3.5
0.2
0.1
7.7
SE
( C)
4.5
5.8
34.3
2.4
3.7
47.3
Fe200
opt3-2
Percentage of
Soluble Sugars
0.2
0.4
3.0
0.1
0.1
0.5
SE
0.9
0.9
2.3
0.3
0.4
2.5
( C)
4.7
6.4
30.7
3.4
4.2
18.0
0.4
0.7
3.9
1.2
1.1
4.6
SE
irt1-1
Percentage of
Soluble Sugars
SE
11
0.4
0.4
3.2
0.4
0.3
8.0
( C)
4.2
5.2
24.5
2.5
3.7
36.7
0.6
0.6
4.7
0.4
0.5
10.9
SE
irt1-1
Percentage of
Soluble Sugars
SE
11
( C)
3.4
4.9
32.3
2.0
2.9
28.4
irt1-1
Percentage of
Soluble Sugars
11
11
11
( C)
3.0
3.9
33.7
1.3
2.0
24.0
SE
11
11
( C)
3.4
4.0
35.0
1.5
2.5
45.0
Fe0
opt3-2
Percentage of
Soluble Sugars
11
0.3
0.3
1.4
0.1
0.1
1.3
( C)
4.2
5.4
37.8
2.4
3.7
26.1
0.7
0.6
0.7
0.5
0.7
2.5
137
Appendix A
Table A3-5. 11C-carbohydrate relocation to belowground and root exudates.
AM
Fe
conditions
Fe0
Fe9
Fe200
Plants
PM
11
11
C transported to
belowground (%)
SE
C transported to
belowground (%)
SE
4.0
5.8
8.2
5.7
5.3
7.0
6.2
6.7
6.6
0.3
0.3
1.4
0.5
0.4
0.4
1.1
0.5
1.3
1.9
4.0
4.5
2.3
2.8
4.6
1.2
2.3
2.1
0.4
0.3
0.7
0.2
0.3
0.6
0.1
0.3
0.2
WT
opt3-2
irt1-1
WT
opt3-2
irt1-1
WT
opt3-2
irt1-1
AM
Fe conditions
Fe0
Fe9
Fe200
Plants
WT
opt3-2
irt1-1
WT
opt3-2
irt1-1
WT
opt3-2
irt1-1
PM
11
C exuded to roots
(%)
2.5
3.3
6.0
4.2
3.5
4.7
4.6
5.6
4.7
11
SE
0.4
0.3
1.2
0.4
0.4
0.5
1.1
0.7
1.3
C exuded to roots
(%)
0.6
0.8
1.1
0.7
0.5
0.7
0.5
0.7
0.8
SE
0.2
0.2
0.3
0.1
0.2
0.2
0.1
0.3
0.2
138
Appendix A
References
1.
Zhang, Y., et al., Application of rhodamine B thiolactone to fluorescence imaging of Hg2+
in Arabidopsis thaliana. Sensors and Actuators B: Chemical, 2011. 153(1): p. 261-265.
2.
Shi, W. and H. Ma, Rhodamine B thiolactone: a simple chemosensor for Hg2+ in aqueous
media. Chem Commun (Camb), 2008(16): p. 1856-8.
139
Appendix B: Protocols
Appendix B
B1. Small scale yeast transformation
I.
Preparation for Competent Cells
1. Inoculate 18 ml of YPD (later 2 ml of 20% sterile glucose is added) with three 3 mm
colonies.
2. Shake media by hand to suspend cells.
3. Incubate at 30 ˚C for 16-18 hr with shaking.
4. Transfer grown cells to 150 ml of YPD.
5. Incubate at 30 ˚C for 3-4 hr.
6. Check OD value at 600 nm (0.5±0.05).
7. Place cells in a 250 ml pre-chilled bottle and centrifuge at 4500 rpm for 5 min at 4 ˚C.
8. Discard the supernatant and suspend cell pellets in 50 ml of sterile ddH2O and shake
by hand.
9. Centrifuge at 4500 rpm for 5 min at 4 ˚C.
10. Decant the supernatant.
11. Resuspend the cell pellet in 50 ml of freshly prepared, sterile 1X TE/LiAc.
12. Centrifuge at 4500 rpm for 5 min at 4 ˚C.
13. Resuspend the cell pellet in 3-5 ml of 1X TE/LiAc.
Note: Keep 4 ˚C from step 7 to 13.
140
Appendix B
II.
Plasmid Transformation
1. Prepare PEG/LiAc solution and plasmids needed to be transformed.
2. Add 100 μl of yeast competent cells into 1.5 ml Eppendorf tubes with ~500 ng of
plasmids. (sometimes DNA carrier is needed)
3. Add 600 μl of 40% PEG/1X TE/LiAc solution and vortex at high speed to mix.
4. Incubate at 30 ˚C for 30 min with shaking.
5. Heat shock for 15 min in a 42 ˚C water bath.
6. Chill cells on ice for 5 min.
7. Centrifuge cells for 5 sec at room temperature.
8. Remove the supernatant.
9. Quick spin down cells and remove the supernatant.
10. Resuspend cells in 500 μl of sterile ddH2O.
11. Spread 100 μl of cells on SD agar medium.
12. Incubate at 30 ˚C for 2-3 days.
YPD: Yeast Extract Peptone Dextrose (commercially available)
SD Medium: Synthetic Defined medium
1X TE/LiAc
10 mM Tris-HCl pH 8.0
1 mM EDTA
0.1 M Lithium acetate
141
Appendix B
PEG/LiAc
40% PEG-4000
0.1 M Lithium actetate
PEG/ 1X TE/LiAc
40% PEG-4000
10 M Tris-HCl pH 8.0
1 mM EDTA
0.1 M Lithium actetate
142
Appendix B
B2. Plasmid DNA Extraction from Yeast for Use in the Transformation
of E. coli
1. Inoculate yeast in 3 ml SD-Ura media and incubate at 30 ˚C with shaking overnight.
2. Centrifuge 1 ml of culture at 14,000 x g for 30 sec at room temperature and remove
all residual liquid.
3. Rinse the cell pellet by 1x TE buffer.
4. Suspend the cells in 100 μl freshly prepared 3% sodium dodecyl sulfate (SDS).
5. Incubate 15 min at room temperature with occasional mixing by several rapid
inversions.
6. Add 500 μl TE buffer. Mix completely by several rapid inversions.
7. Add 60 μl 3 M sodium acetate. Mix completely by several rapid inversions.
8. Add 600 μl phenol:chloroform:isoamyl alcohol, 25:24:1.
9. Vortex for 2 min at full speed.
10. Centrifuge in a microcentrifuge at 14,000 x g for 2 min. Transfer the upper aqueous
phase to a new microcentrifuge tube.
11. Repeat steps 8-10.
12. Add 1 ml of 100% ethanol. Mix completely by several rapid inversions.
13. Incubate at -80 ˚C for at least 20 min.
14. Centrifuge in a microcentrifuge at 14,000 x g for 20 min.
15. Add 100 μl of 70% ethanol and incubate for 1 hr.
16. Centrifuge in a microcentrifuge at 14,000 x g for 5 min.
17. Discard the supernatant. Centrifuge for 10 sec and remove all residual supernatant.
18. Suspend the pellet in 50 μl H2O.
143
Appendix B
TE buffer
10 mM Tris-HCl pH 8.0
1 mM EDTA
144
3
1.5
1.5
2
1
1
246.47
80
61.83
169.02
287.56
249.7
241.95
136.1
246.47g/L
20g/0.25L
2.86g/L
1.54g/L
0.22g/L
0.051g/L
0.12g/L
68g/0.5L
49.3g
16g
0.57g
0.31g
0.044g
0.01g
0.024g
27.2g
NH4NO3
Micro nutrients
H3BO3
MnSO4.H2O
ZnSO4.7H2O
CuSO4.5H2O
NaMoO4
KH2PO4
0.009M
0.00077M
0.0002M
0.0005M
0.046M
(pH to 6 w/ NaOH)
1M
1M
MgSO4
1M
0.75
3.75
2.5
236.15
236g/0.5L
94.4g
Ca(NO3)2. 4H2O
2M
0.5
1.5 Li
3.75
1 Li (ml)
2.5
Compound
KNO3
Stock conc.
2M
Stock prep. In 200ml Stock prep. in 1Li
202g/L
40.4g
FW
101.11
Hoagland's solution (w/o Fe)
Table B3-1. Modified Hoagland’s solution (without Fe)
Appendix B
B3. Modified Hoagland’s solution (without Fe)
145
VITA
Lihui Song was born and raised in Tianjin, China. She received her bachelor’s
degree in Applied Chemistry from the Tianjin University (Tianjin, China) in May 2008.
To extend her knowledge and experience, Lihui chose to pursuit a further education in
US. She was accepted as a Ph.D. student in the Chemistry Department at University of
Missouri where she joined Jurisson research group and earned her Ph.D. degree in 2013.
In the same year, she received the Mizzou Advantage Prepare for Future Faculty
fellowship from University of Missouri, specifically in the One Health/One Medicine
area. During the fellowship, she will work with Dr. Scott Frey in the MU Brain Imaging
Center (BIC) on Magnetic Resonance Spectroscopy (MRS) studies on human brains, as
well as teach several courses related to her research.
146